Inhibitor of IGFBP3/TMEM219 axis and diabetes

ABSTRACT

The present invention relates to the role of the IGFBP3/TMEM219 axis in the onset of diabetes and the related use of IGFBP3/TMEM219 axis inhibitors for the treatment and/or prevention of diabetes. The invention also relates to a method to identify a subject at risk of developing Type 1 and/or Type 2 diabetes and relative kit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/831,235, filed Dec. 4, 2017, which is a 371 National Stage Entry ofInternational Application No. PCT/EP2016/062792, filed Jun. 6, 2016,which claims the benefit of European Patent Application No. 16169222.3,filed May 11, 2016, and European Patent Application No. 15170679.3,filed Jun. 4, 2015, the contents of each of which are each incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 4, 2018, isnamed 40291US_CRF_sequencelisting.txt and is 8,192 bytes in size.

TECHNICAL FIELD

The present invention relates to the role of the IGFBP3/TMEM219 axis inthe onset of diabetes and the related use of IGFBP3/TMEM219 axisinhibitors for the treatment and/or prevention of diabetes. Theinvention also relates to a method to identify a subject at risk ofdeveloping Type 1 and/or Type 2 diabetes and relative kit.

BACKGROUND ART

Gastrointestinal disorders, consisting of gastroparesis, abdominaldistension, irritable bowel syndrome and fecal incontinence, are commonin individuals with type 1 diabetes (T1D)(1993). Indeed up to 80% ofindividuals with long-standing T1D, who are generally affected byseveral diabetic complications including end stage renal disease(ESRD)(1993; Atkinson et al., 2013; Fiorina et al., 2001), showintestinal symptoms. The presence of these gastrointestinal symptoms,known as diabetic enteropathy (DE), significantly reduces the quality oflife (1993; Atkinson et al., 2013; Camilleri, 2007; Talley et al., 2001)and has a largely unknown pathogenesis (Feldman and Schiller, 1983).Preclinical studies showed significant derangement of the intestinalmucosa morphology in diabetic rodents (Domenech et al., 2011; Zhao etal., 2003), suggesting that in T1D intestinal homeostasis may bealtered; however, little data are available in humans. The intestinalepithelium is maintained by intestinal stem cells and their niche, whichrespond to physiological stress and to environmental injury (Barker,2014; Medema and Vermeulen, 2011). Colonic stem cells (CoSCs), locatedat the crypt base of the large intestine and expressing the ephrin Breceptor 2 (EphB2), leucine-rich repeat containing G protein-coupledreceptor 5 (LGR5), h-TERT and aldehyde dehydrogenase (Aldh), among othermarkers (Carlone and Breault, 2012; Carpentino et al., 2009; Jung etal., 2011; Sato and Clevers, 2013), constitute with the localmicroenvironment the CoSC niche (van der Flier and Clevers, 2009; Zekiet al., 2011). Recent studies have established conditions thatrecapitulate many features of intestinal homeostasis and generate normalself-renewing large crypt organoids in vitro, or so-called “mini-guts”(Sato and Clevers, 2013). Whether systemic factors, such as circulatinghormones, serve to control the CoSCs remains to be established (Stangeand Clevers, 2013).

The treatment of gastrointestinal disorders, in particular diabeticenteropathy includes symptomatic drugs and reliever medications fordiarrhea, abdominal pain, constipation, and dyspepsia. Up to date thereis no specific treatment available for diabetic enteropathy.

The diagnosis of gastrointestinal disorders, in particular diabeticenteropathy includes colon endoscopy, gastric endoscopy, anorectalmanometry, esophageal manometry and analysis of fecal samples,evaluation of peripheral cancer markers (i.e. CEA, Ca 19.9,alpha-fetoprotein, Ca125) and of celiac markers. None of theaforementioned method is capable of providing a certain diagnosis ofdiabetic enteropathy.

WO 2011133886 and WO2007024715 disclose a therapeutic composite in theform of a IGFBP3 binding antibody.

WO0187238 relates to an anticancer pharmaceutical composition comprisinga therapeutically effective TMEM219, in particular for the treatment ofcolon cancer.

WO 2014089262 discloses the use of IGFBP3 as a marker of diagnosis ofchronic inflammation (obesity) disorders (in particular, inflammatorybowel disease such as UC and Crohn's disease and colon cancer).

U.S. Pat. No. 6,066,464 relates to an immunoassay for the detection ofIGFBP3 on a solid support that is paper.

WO2013152989 relates to the use of IGFBP3 as a biomarker of colorectalcancer.

WO0153837 discloses a method of monitoring or diagnosing diseaseconditions that involve measuring a combination of tumor markers and atleast one component of the IGF axis. IGFBP3 is proposed as a marker ofcolon tumors.

Type 1 diabetes (T1D) has historically been regarded as a Tcell-mediated autoimmune disease, resulting in the destruction ofinsulin-producing pancreatic beta cells (Bluestone et al., 2010;Eisenbarth, 1986). According to this perspective, an initiating factortriggers the immune response against autoantigens, and the subsequentnewly activated autoreactive T cells target and further destroy thepancreatic islets and insulin-producing beta cells (Bluestone et al.,2010). Whether destruction of beta cells is solely determined by theautoimmune attack or whether other mechanisms such as paracrinemodulation, metabolic deregulation, non-immune beta cell apoptosis andhalted beta cell regeneration contribute to T1D pathogenesis is now amatter of debate (Atkinson and Chervonsky, 2012; Atkinson et al., 2015).Recently, it has been observed that environmental factors are requiredto initiate the autoimmune response in T1D, particularly viralinfections (Filippi and von Herrath, 2008), and studies of the impact ofgut microbiota have revealed that enteroviruses are involved inactivating autoreactive T cells (McLean et al., 2015). Ongoing studiesare also focused on other environmental risk factors such as diet,neonatal exposure to milk and gluten, and age at weaning, suggestingthat a new approach to study the pathogenesis of T1D is graduallyemerging (McLean et al., 2015), such that genetic factors are no longerconsidered to be the sole determinant of T1D (Alper et al., 2006),(Oilinki et al., 2012).

Moreover, the efficacy of immunotherapeutic strategies, which have beenconsidered in the last decade to be the principal prospect forestablishing a cure for T1D, is now being questioned (Ben Nasr et al.,2015a). While targeting the autoimmune response using animmunosuppressive treatment or a pro-regulatory regimen was shown to besatisfactory in rodents, such strategies conversely achieved insulinindependence in a negligible number of T1D individuals (Atkinson et al.,2015). In addition to underscoring the difference between animal modelsand humans, these data also shed light on the fact that investigation ofthe immune response primarily examined immune events occurring in theperiphery, while little is known with respect to the disease processthat occurs within islets and particularly in beta cells. In thisregard, the discovery of novel factors involved in theinitiation/facilitation of beta cell loss in T1D will be of significantvalue. Such discoveries may pave the way for novel therapeuticapproaches capable of halting or delaying the very first phase of thedisease. Then, there is still the need for alternative treatment for T1Dand T2D.

WO2008153788 claims a method to inhibit or reduce IGFBP3 levels to treatinsulin resistance or TD2, wherein the inhibitor is a nucleic acidcomplementary to IGFBP3 mRNA or an antibody that binds IGFBP3, antiIGFBP-3. The document is silent about the IGFBP3/TMEM219 axis.

Muzumdar et al. (Muzumdar et al., 2006) discloses that IGFBP3 acts as aninsulin antagonist through a central mechanism leading to a reducedperipheral glucose uptake. This document does not disclose theinhibition of the IGFBP3/TMEM219 axis.

WO9739032 claims the use of an IGFBP3 inhibitor to treat diabetes,wherein the inhibitor prevents IGFBP-3 binding to IGF-1. Inhibition ofIGFBP3/TMEM219 axis is not contemplated.

D'Addio et al., (2015) indicates that eco-TEM219 normalize circulatingIGF-I/IGFBP3 levels.

WO2007024715 relates to the use of engineered multivalent andmultispecific binding proteins, namely dual variable domainimmunoglobulins, which bind two different antigens or target peptidesusing a single middle linker and are bispecific. The document mentionsamong the numerous target proteins, IGFBP3 in combination with othermembers of the family.

WO2011133886: relates to a method of generating antibodies and othermultimeric protein complexes, namely heteromutlimeric proteins, capableof specifically binding to more than one target. IGFBP3 may represent apotential target.

SUMMARY OF THE INVENTION

Whether systemic factors serve to control the homeostasis of colonicepithelium and of colonic stem cells (CoSCs) remains unclear. Theinventors hypothesize that a circulating “hormonal” dyad controls CoSCsand is disrupted in long-standing type 1 diabetes (T1D) leading todiabetic enteropathy (DE). Individuals with long-standing T1D exhibitedabnormalities of intestinal mucosa and CoSCs, and failure to generate invitro mini-guts. Serum proteomic profiling revealed altered circulatinglevels of insulin-like growth factor 1 (IGF-I) and its binding protein-3(IGFBP3) in long-standing T1D individuals, with evidences of anincreased hyperglycemia-mediated IGFBP3 hepatic release. IGFBP3prevented mini-gut growth in vitro via aTMEM219-dependent/caspase-mediated IGF-I-independent effect anddisrupted CoSCs in preclinical models in vivo. The restoration ofnormoglycemia in long-standing T1D, with kidney-pancreastransplantation, and the treatment with an ecto-TMEM219 recombinantprotein in diabetic mice, re-established CoSCs by restoring appropriateIGF-I/IGFBP3 circulating levels. The peripheral IGF-I/IGFBP3 dyadcontrols CoSCs and is dysfunctional in DE.

Here the inventors demonstrate that individuals with long-standing T1Dand DE have altered CoSCs and show increased levels of IGFBP3.Administration of IGFBP3 alters CoSC regenerative properties and mucosamorphology in vitro and in vivo, in a preclinical model of DE, byquenching circulating IGF-I and by exerting aTMEM219-dependent/caspase-mediated toxic effect on CoSCs. Finally, a newecto-TMEM219 recombinant protein, based on the extracellular domain ofthe IGFBP3 receptor (TMEM219) was generated. ecto-TMEM219 quenchesperipheral IGFBP3 and prevents its binding to IGFBP3 receptor, TMEM219.Then, targeting IGFBP3 with such ecto-TMEM219 recombinant protein,expressed on CoSCs, abrogates IGFBP3 deleterious effects in vitro and invivo.

The present invention reports compelling data showing that IGFBP3release is increased in individuals at high-risk for T1D and T2D.Interestingly, the inventors have discovered that the IGFBP3 receptor,TMEM219, is expressed in a beta cell line and on murine/human islets,and that its ligation by IGFBP3 is toxic to beta cells, raising thepossibility of the existence of an endogenous beta cell toxin. Thissuggests that beta cell toxin(s) [betatoxin(s)] may be involved in thepathogenesis of TD1, in particular in the early phase, when islet/betacell injuries may facilitate the exposure of autoantigens to immunecells, thus creating a local inflamed environment and a sustained immunereaction. Interestingly, authors have observed elevated levels of IGFBP3in pre-T2D and in T2D individuals as well, suggesting that a potentialrole for this axis is also evident in T2D.

The inventors have also observed that IGFBP3 may induce apoptosis ofbeta cells and of murine/human islets in vitro in a caspase 8-dependentmanner. Finally, the newly generated recombinant ecto-TMEM219 protein,based on the TMEM219 extracellular domain, capable of quenching IGFBP3,prevents its signaling via TMEM219 on pancreatic beta cells.Ecto-TMEM219 treatment reduces beta cell loss, improves islet insulincontent and glycometabolic control in murine models of diabetes (T1D andT2D) in vivo, while in vitro it protects islets and beta cells fromIGFBP3-induced apoptosis. The inventors demonstrate that IGFBP3 is anendogenous peripheral beta cell toxin (or betatoxin) that isincreasingly released in individuals at high-risk for diabetes (T1D andT2D). Concomitant expression of the IGFBP3 receptor (TMEM219) on betacells initiates/facilitates beta cell death, thus favoring diabetesonset/progression.

In other words, the invention is based on the finding that TMEM219, theIGFBP3 receptor that mediates IGFBP3/IGF1 independent detrimentaleffects, is expressed on pancreatic islets and beta cells; moreover,targeting the IGFBP3/TMEM219 axis with ecto-TMEM219 re-establishesappropriate IGFBP3 signaling in diabetic mice and prevents beta cellloss and preserves islet morphology, thereby confirming the criticalrole of the IGFBP3/TMEM219 axis in favoring beta cell loss in diabetes.

The present therapeutic approach, based on the inhibition ofIGFBP3/TMEM219 axis, may overcome the limits of the current therapiesfor T1D and T2D as it could prevent the beta cell damage and theconsequent reduced or abolished insulin secretion that leads to thedevelopment of diabetes.

Then, the advantages of the present invention over prior art treatmentsare:

-   -   Prevention of beta cell and islets destruction    -   Protection of beta cell mass and of insulin-producing cells    -   Prevention of major diabetes complications    -   Limitation of autoimmune attack towards pancreatic islets in T1D    -   Prevention of insulin resistance in T2D and    -   No requirement for immunotherapy in T1D.

Then the invention provides an inhibitor of IGFBP3/TMEM219 axis for usein the treatment and/or prevention of diabetes in a subject.

Preferably said inhibitor is selected from the group consisting of:

-   -   a) a polypeptide;    -   b) a polynucleotide coding for said polypeptide or a        polynucleotide able to inhibit IGFBP3/TMEM219 axis;    -   c) a vector comprising or expressing said polynucleotide;    -   d) a host cell genetically engineered expressing said        polypeptide or said polynucleotide;    -   e) a small molecule;    -   f) a peptide, a protein, an antibody, an antisense        oligonucleotide, a siRNA, antisense expression vector or        recombinant virus or any other agent able to inhibit or        IGFBP3/TMEM219 axis.

Preferably said inhibitor is the receptor TMEM219 or a fragment thereof.

Preferably the fragment of TMEM219 is a fragment comprising anextracellular domain of TMEM219.

In a preferred embodiment the inhibitor is ecto-TMEM219. Preferably theinhibitor is soluble.

Preferably said inhibitor is a fusion protein TMEM219-Ig, preferablysaid fusion protein quenches circulating IGFBP3 and prevents its bindingto TMEM219.

Preferably the inhibitor is an anti-IGFBP3 antibody, preferably saidantibody selectively blocks the TMEM219-binding site;

Preferably said inhibitor is an anti-TMEM219 antibody, preferably saidantibody occupies the IGFBP3 binding site of TMEM219 receptor thuspreventing IGFBP3 binding.

More preferably said inhibitor is an oligonucleotide complementary toIGFBP3 mRNA.

In a preferred embodiment the diabetes is Type-1 or Type-2 diabetes.

Still preferably the subject is selected from the group consisting of: asubject at risk of developing Type-1 and/or Type-2 diabetes, a subjectwith early stage Type-1 and/or Type-2 diabetes.

The present invention also provides a pharmaceutical composition for usein the treatment and/or prevention of diabetes comprising the inhibitorof the invention and pharmaceutically acceptable carriers. Preferablythe pharmaceutical composition further comprises a therapeutic agent.

Preferably the therapeutic agent is selected from the group consistingof: insulin in any form, Pramlintide (Symlin), angiotensin-convertingenzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs),Aspirin, Cholesterol-lowering drugs. Metformin (Glucophage, Glumetza,others), Sulfonylureas (glyburide (DiaBeta, Glynase), glipizide(Glucotrol) and glimepiride (Amaryl), Meglitinides (for instancerepaglinide (Prandin) and nateglinide (Starlix)), Thiazolidinediones(Rosiglitazone (Avandia) and pioglitazone (Actos) for examples), DPP-4inhibitors (sitagliptin (Januvia), saxagliptin (Onglyza) and linagliptin(Tradjenta)), GLP-1 receptor agonists (Exenatide (Byetta) andliraglutide (Victoza)), SGLT2 inhibitors, examples include canagliflozin(Invokana) and dapagliflozin (Farxiga).

The present invention also provides a method to identify a subject atrisk of developing Type-1 and/or Type-2 or to monitor the response to atherapeutic treatment in a subject comprising:

-   -   a) measuring the amount of the protein IGFBP3 or the amount of        the polynucleotide coding for said protein in a biological        sample obtained from the subject;    -   b) comparing the measured quantity of the protein IGFBP3 or        measured quantity of the polynucleotide coding for said protein        to a control amount, wherein if the measured quantity is higher        than the control amount, the subject is at risk of developing        Type-1 and/or Type-2 diabetes.

Preferably the quantity of IGFBP3 is measured by an antibody.

More preferably the biological sample is selected from the groupconsisting of: serum, urine, cell culture supernatant.

The present invention also provides a kit comprising means to measurethe amount of the protein IGFBP3 and/or means to measure the amount ofthe polynucleotide coding for said protein and optionally, control meansfor use in the method of the invention.

In the present invention inhibiting the IGFBP3/TMEM219 axis meansblocking IGFBP3 binding to TMEM219, for instance by quenching IGFBP3from the circulation, it also means blocking the IGFBP3-binding site ofTMEM219, blocking IGFBP3 binding site on TMEM219. It further meansinhibiting TMEM219 function and/or expression and/or signaling, this maybe achieved for instance by silencing TMEM219 expression, in particularwith SiRNA or oligonucleotides. It also means inhibiting the functionand/or expression of IGFBP3.

According to the invention, an inhibitor of IGFBP3 binding to TMEM219can be one of the following molecules:

-   -   Soluble Ecto-TMEM219 (extracellular portion of TMEM219) which        neutralizes circulating IGFBP3;    -   Fusion protein TMEM219-Ig, a Fc-based fusion protein composed of        an immunoglobulin Fc domain that is directly linked to TMEM219        peptide or to its extracellular portion, which quenches        circulating IGFBP3 and prevents its binding to TMEM219 expressed        on beta cells;    -   Anti-IGFBP3 antibody that selectively blocks the TMEM219-binding        site;    -   Anti-TMEM219 antibody, which occupies the IGFBP3 binding site of        TMEM219 receptor thus preventing IGFBP3 binding (having        antagonistic activity with respect to IGFBP3)    -   Oligonucleotides complementary to IGFBP3 mRNA

In the present invention the patient that may be treated are individualswho are at risk for developing T1D (autoimmune diabetes, based on thepresence of peripheral anti-islet autoantibodies or geneticpredisposition or familiar predisposition or altered beta cell function)or T2D (non autoimmune diabetes based on the evidence of an impairedfasting glucose and/or impaired glucose tolerance without fulfilling thecriteria for the diagnosis of diabetes), or individuals who develop T1Dor T2D in any stage of the disease, in particular a subject with earlystage Type-1 and/or Type-2 diabetes, with the purpose of protecting betacells from further destruction. The presence of any degree of preservedbeta cells is the only requirement for assessing the successful therapy.

The expression of IGFBP3 may be measured by means of RT-PCR on tissuesand cells, Western blot on tissues and cells, Immunohistochemistry ontissues, Immunofluorescence on tissue and cells. Levels of IGFBP3 inbiological fluids can be measured by immune-targeted assays andproteomic analysis.

The function of IGFBP3 may be measured by means of detecting Caspases 8and 9 expression on target cells using RT-PCR, microarrays, byco-culturing target cells/structures with Pan Caspase inhibitor,Caspases 8 and 9 inhibitors and measuring live cells/structures.

In the present invention “inhibit or block the interaction of IGFBP3with its receptor TMEM219” means quenching circulating IGFBP3 andpreventing its binding to TMEM219 receptor expressed on pancreaticislets and beta cells. The IGFBP3-TMEM219 binding could be preventedalso by the use of an IGFBP3-blocking antibody. In addition, a TMEM219blocking antibody could bind TMEM219 receptor thus rendering thereceptor unavailable when IGFBP3 comes from the circulation.

The inhibitor of the invention may be the receptor TMEM219(MGNCQAGHNLHLCLAHHPPLVCATLILLLLGLSGLGLGSFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSRLLVLGSFLLLFCGLLCCVTAMCFHP RRESHWSRTRL,SEQ ID NO: 1) or a fragment thereof.

In particular the fragment of TMEM219 is designed such as toblock/prevent IGFBP3 access and/or binding to TMEM219, it has a smallermolecular weight, it contains five cysteins that form disulfide bridgesand a globular structure. Preferably the fragment is at least 50 aminoacid long, preferably 100 amino acid long, still preferably 120 aminoacid long, yet preferably 150 amino acid long, preferably at least 160amino acid long.

In a preferred embodiment the fragment is at least 162, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 amino acidlong. Preferably the fragment has at least 65% identity with thesequence of TMEM219, preferably at least 70%, 75%, 80%, 85%, 90%, 95% or99% identity with the sequence of TMEM219.

Preferably the fragment of TMEM219 is a fragment of an extracellulardomain of TMEM219 (ecto-TMEM219), in particular the fragment comprisesthe sequence:

(SEQ ID No. 2) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKL LTSEELALCGSR.

Preferably the fragment of TMEM219 is an extracellular domain ofTMEM219, in particular the fragment comprises the sequence:

(SEQ ID No. 3) SFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLV LTKLLTSEELALCGSR

Preferably the fragment of TMEM219 consists of:

(SEQ ID No. 2) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKL LTSEELALCGSR.

Preferably the fragment of TMEM219 consists of:

(SEQ ID No. 3) SFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLV LTKLLTSEELALCGSR.

In the present invention TMEM219 is preferably eukaryote TMEM219,preferably a mammal TMEM219, still preferably human TMEM219.

The interaction of IGFBP3 with TMEM219 may be measured by means ofindirect assessment of the effects of IGFBP3 on target cells (increasedCaspase 8 and 9 expression with RT-PCR), direct assessment ofIGFBP3-IGFBP3-receptor (TMEM219) binding with Liquid or Solid PhaseLigand Binding Assays (i.e. immunoprecipitation, RT-PCR, immunoassays)and Non-radioactive Ligand Binding Assays.

In the present invention “long-standing T1D” means a history of type 1diabetes longer than 15 years associated with the development ofdiabetic complications.

In a preferred aspect of the invention, the inhibitor is an antibody orsynthetic or recombinant derivative thereof. Said antibody is preferablya monoclonal or polyclonal antibody, or synthetic or recombinantderivatives thereof, more preferably said antibody being a humanizedmonoclonal antibody.

Preferably, said polynucleotide is a RNA or DNA, preferably a siRNA, ashRNA, a microRNA or an antisense oligonucleotide.

In a preferred embodiment, the above vector is an expression vectorselected from the group consisting of: plasmids, viral particles andphages.

Preferably, said host cell is selected from the group consisting of:bacterial cells, fungal cells, insect cells, animal cells, plant cells,preferably being an animal cell, more preferably a human cell.

In a preferred embodiment, the inhibitor as above defined (a) iscombined with at least one therapeutic agent (b) to define a combinationor combined preparation. The therapeutic agent may be an anti-diabeticagent, an agent used to prevent diabetes, an anti-apoptotic agent, ananti-inflammatory agent, immune suppressive agent, adjuvant therapy inorgan transplantation, protective agent in cell therapy approach a painreliever.

Examples of therapeutic agent is insulin therapy, in any form,Pramlintide (Symlin), angiotensin-converting enzyme (ACE) inhibitors orangiotensin II receptor blockers (ARBs), Aspirin, Cholesterol-loweringdrugs. Metformin (Glucophage, Glumetza, others), Sulfonylureas(glyburide (DiaBeta, Glynase), glipizide (Glucotrol) and glimepiride(Amaryl), Meglitinides (for instance repaglinide (Prandin) andnateglinide (Starlix)), Thiazolidinediones (Rosiglitazone (Avandia) andpioglitazone (Actos) for examples), DPP-4 inhibitors (sitagliptin(Januvia), saxagliptin (Onglyza) and linagliptin (Tradjenta)), GLP-1receptor agonists (Exenatide (Byetta) and liraglutide (Victoza)), SGLT2inhibitors, examples include canagliflozin (Invokana) and dapagliflozin(Farxiga).

The terms “combination” and “combined preparation” as used herein alsodefine a “kit of parts” in the sense that the combination partners (a)and (b) as defined above can be dosed independently or by use ofdifferent fixed combinations with distinguished amounts of thecombination partners (a) and (b), i.e. simultaneously or at differenttime points. The parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partner (a) to the combination partner (b) to beadministered in the combined preparation can be varied, e.g. in order tocope with the needs of a patient sub-population to be treated or theneeds of the single.

The combination therapy may result in unexpected improvement in thetreatment of diabetes. When administered simultaneously, sequentially orseparately, the inhibitor and the other therapeutic agent may interactin a synergistic manner to reduce diabetes. This unexpected synergyallows a reduction in the dose required of each compound, leading to areduction in the side effects and enhancement of the clinicaleffectiveness of the compounds and treatment. Determining a synergisticinteraction between one or more components, the optimum range for theeffect and absolute dose ranges of each component for the effect may bedefinitively measured by administration of the components over differentw/w ratio ranges and doses to patients in need of treatment. For humans,the complexity and cost of carrying out clinical studies on patientsrenders impractical the use of this form of testing as a primary modelfor synergy. However, the observation of synergy in one species can bepredictive of the effect in other species and animal models exist, asdescribed herein, to measure a synergistic effect and the results ofsuch studies can also be used to predict effective dose and plasmaconcentration ratio ranges and the absolute doses and plasmaconcentrations required in other species by the application ofpharmacokinetic/pharmacodynamic methods. Established correlationsbetween diabetes models and effects seen in man suggest that synergy inanimals may e.g. be demonstrated in the models as described in theExamples below.

The above pharmaceutical compositions are preferably for systemic, oral,locally, preferably rectally, or topical administration.

Control amount is the amount measured in a proper control.

Control means can be used to compare the amount or the increase ofamount of the compound as above defined to a proper control. The propercontrol may be obtained for example, with reference to known standard,either from a normal subject or from normal population.

The above diagnosis method may also comprise a step of treating thesubject, in particular the treatment may be an inhibitor ofIGFBP3/TMEM219 axis as defined in the present invention or an existingtreatment for diabetes such as indicated above.

The means to measure the amount of IGFBP3 as above defined arepreferably at least one antibody, functional analogous or derivativesthereof. Said antibody, functional analogous or derivatives thereof arespecific for said compound.

In a preferred embodiment, the kit of the invention comprises:

-   -   a solid phase adhered antibody specific for said compound;    -   detection means of the ligand specific-biomarker complex.

The kits according to the invention can further comprise customaryauxiliaries, such as buffers, carriers, markers, etc. and/orinstructions for use.

The proper control may be a sample taken from a healthy patient or froma patient affected by a disorder other than diabetes.

In the case of a method or a kit for monitoring the progression of thediabetes, the progress of the disease is monitored and the propercontrol may be a sample taken from the same subject at various times orfrom another patient, and the proper control amount may by the amount ofthe same protein or polynucleotide measured in a sample taken from thesame subject at various times or from another patient.

In the case of a method or a kit for monitoring the efficacy or responseto a therapeutic treatment, the proper control may by a sample takenfrom the same subject before initiation of the therapy or taken atvarious times during the course of the therapy and the proper controlamount may be the amount of the same protein or polynucleotide measuredin a sample taken from the same subject before initiation of the therapyor taken at various times during the course of the therapy. The therapymay be the therapy with the inhibitor of the present invention.

In the present invention, the expression “measuring the amount” can beintended as measuring the amount or concentration or level of therespective protein and/or mRNA thereof and/or DNA thereof, preferablysemi-quantitative or quantitative. Measurement of a protein can beperformed directly or indirectly. Direct measurement refers to theamount or concentration measure of the biomarker, based on a signalobtained directly from the protein, and which is directly correlatedwith the number of protein molecules present in the sample. Thissignal—which can also be referred to as intensity signal—can beobtained, for example, by measuring an intensity value of a chemical orphysical property of the biomarker. Indirect measurements include themeasurement obtained from a secondary component (e.g., a differentcomponent from the gene expression product) and a biological measurementsystem (e.g. the measurement of cellular responses, ligands, “tags” orenzymatic reaction products).

The term “amount”, as used in the description refers but is not limitedto the absolute or relative amount of proteins and/or mRNA thereofand/or DNA thereof, and any other value or parameter associated with thesame or which may result from these. Such values or parameters compriseintensity values of the signal obtained from either physical or chemicalproperties of the protein, obtained by direct measurement, for example,intensity values in an immunoassay, mass spectroscopy or a nuclearmagnetic resonance. Additionally, these values or parameters includethose obtained by indirect measurement, for example, any of themeasurement systems described herein. Methods of measuring mRNA and DNAin samples are known in the art. To measure nucleic acid levels, thecells in a test sample can be lysed, and the levels of mRNA in thelysates or in RNA purified or semi-purified from lysates can be measuredby any variety of methods familiar to those in the art. Such methodsinclude hybridization assays using detectably labeled DNA or RNA probes(i.e., Northern blotting) or quantitative or semi-quantitative RT-PCRmethodologies using appropriate oligonucleotide primers. Alternatively,quantitative or semi-quantitative in situ hybridization assays can becarried out using, for example, tissue sections, or unlysed cellsuspensions, and detectably labeled (e.g., fluorescent, orenzyme-labeled) DNA or RNA probes. Additional methods for quantifyingmRNA include RNA protection assay (RPA), cDNA and oligonucleotidemicroarrays, representation difference analysis (RDA), differentialdisplay, EST sequence analysis, and serial analysis of gene expression(SAGE).

If by comparing the measured amount of the protein IGFBP3 or of thepolynucleotide coding for said protein with the amount obtained from acontrol sample, the amount of said compound in the sample isolated fromthe subject corresponds to a higher value, the subject may present thedisease or go towards an aggravation of said disease.

If by comparing the measured amount of the protein IGFBP3 or of thepolynucleotide coding for said protein with the amount obtained from acontrol sample, the amount of said compound in the sample isolated fromthe subject corresponds to a similar or lower value, the subject may benot affected by the disease or go toward an amelioration of the disease,respectively.

Alternatively, the expression “detection” or “measuring the amount” isintended as measuring the alteration of the molecule. Said alterationcan reflect an increase or a decrease in the amount of the compounds asabove defined. An increase of the protein IGFBP3 or of thepolynucleotide coding for said protein can be correlated to anaggravation of the disease. A decrease the protein IGFBP3 or of thepolynucleotide coding for said protein can be correlated to anamelioration of the disease or to recovery of the subject.

The expression “protein IGFBP3” or “IGFBP3” or “TMEM219” is intended toinclude also the corresponding protein encoded from a IGFBP3 or TMEMorthologous or homologous genes, functional mutants, functionalderivatives, functional fragments or analogues, isoforms thereof.

The expression “gene IGFBP3” or “IGFBP3” or “gene TMEM219” or “TMEM219”is intended to include also the corresponding orthologous or homologousgenes, functional mutants, functional derivatives, functional fragmentsor analogues, isoforms thereof.

In the present invention “functional mutants” of the protein are mutantsthat may be generated by mutating one or more amino acids in theirsequences and that maintain their activity for the treatment ofdiabetes. Indeed, the protein of the invention, if required, can bemodified in vitro and/or in vivo, for example by glycosylation,myristoylation, amidation, carboxylation or phosphorylation, and may beobtained, for example, by synthetic or recombinant techniques known inthe art. The protein of the invention “IGFBP3” or “TMEM219” may bemodified to increase its bioavailability or half-life by know method inthe art. For instance, the protein may be conjugated to a polymer, maybe pegylated etc.

In the present invention the active ingredients may also be entrapped inmicrocapsule prepared, for example, by coacervation techniques or byinterfacial polymerization, for example,

hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin

microspheres, microemulsions, nano-particles and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma]ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate,and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods. When encapsulated antibodies remain in the bodyfor a long time, they may denature or aggregate as a result of exposureto moisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

In the present invention “functional” is intended for example as“maintaining their activity” e.g. therapeutic treatment of diabetes.

The term “analogue” as used herein referring to a protein means amodified peptide wherein one or more amino acid residues of the peptidehave been substituted by other amino acid residues and/or wherein one ormore amino acid residues have been deleted from the peptide and/orwherein one or more amino acid residues have been deleted from thepeptide and or wherein one or more amino acid residues have been addedto the peptide. Such addition or deletion of amino acid residues cantake place at the N-terminal of the peptide and/or at the C-terminal ofthe peptide.

The term “derivative” as used herein in relation to a protein means achemically modified peptide or an analogue thereof, wherein at least onesubstituent is not present in the unmodified peptide or an analoguethereof, i.e. a peptide which has been covalently modified. Typicalmodifications are amides, carbohydrates, alkyl groups, acyl groups,esters and the like. As used herein, the term “derivatives” also refersto longer or shorter polypeptides having e.g. a percentage of identityof at least 41%, preferably at least 41.5%, 50%, 54.9%, 60%, 61.2%,64.1%, 65%, 70% or 75%, more preferably of at least 85%, as an exampleof at least 90%, and even more preferably of at least 95% with IGFBP3,or with an amino acid sequence of the correspondent region encoded froma IGFBP3 orthologous or homologous gene.

As used herein “fragments” refers to polypeptides having preferably alength of at least 10 amino acids, more preferably at least 15, at least17 amino acids or at least 20 amino acids, even more preferably at least25 amino acids or at least 37 or 40 amino acids, and more preferably ofat least 50, or 100, or 150 or 200 or 250 or 300 or 350 or 400 or 450 or500 amino acids.

According to the present invention, an “effective amount” of acomposition is one that is sufficient to achieve a desired biologicaleffect, in this case an amelioration or the treatment of diabetes.

It is understood that the effective dosage will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The provided ranges of effective doses of the inhibitor ormolecule of the invention (e.g. from 1 mg/kg to 1000 mg/kg, inparticular systemically administered) are not intended to limit theinvention and represent preferred dose ranges. However, the preferreddosage can be tailored to the individual subject, as is understood anddeterminable by one of skill in the art, without undue experimentation.

The administration of oligonucleotides of the present invention may becarried out by known methods, wherein a nucleic acid is introduced intoa desired target cell in vitro or in vivo.

An aspect of the present invention comprises a nucleic acid constructcomprised within a delivery vehicle. A delivery vehicle is an entitywhereby a nucleotide sequence can be transported from at least one mediato another. Delivery vehicles may be generally used for expression ofthe sequences encoded within the nucleic acid construct and/or for theintracellular delivery of the construct. It is within the scope of thepresent invention that the delivery vehicle may be a vehicle selectedfrom the group of RNA based vehicles, DNA based vehicles/vectors, lipidbased vehicles, virally based vehicles and cell based vehicles. Examplesof such delivery vehicles include: biodegradable polymer microspheres,lipid based formulations such as liposome carriers, coating theconstruct onto colloidal gold particles, lipopolysaccharides,polypeptides, polysaccharides, pegylation of viral vehicles.

In one embodiment of the present invention may comprise a virus as adelivery vehicle, where the virus may be selected from: adenoviruses,retroviruses, lentiviruses, adeno-associated viruses, herpesviruses,vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki forestvirus, poxviruses, RNA virus vector and DNA virus vector. Such viralvectors are well known in the art.

Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, transfection, electroporation and microinjection and viralmethods. Another technique for the introduction of DNA into cells is theuse of cationic liposomes. Commercially available cationic lipidformulations are e.g. Tfx 50 (Promega) or Lipofectamin 2000 (LifeTechnologies).

The compositions of the present invention may be in form of a solution,e.g. an injectable solution, a cream, ointment, tablet, suspension orthe like. The composition may be administered in any suitable way, e.g.by injection, particularly by intraocular injection, by oral, topical,nasal, rectal application etc. The carrier may be any suitablepharmaceutical carrier. Preferably, a carrier is used, which is capableof increasing the efficacy of the RNA molecules to enter thetarget-cells. Suitable examples of such carriers are liposomes,particularly cationic liposomes.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The recombinant expression vectors of the invention can beprepared using standard recombinant DNA techniques. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from CoIE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based. The recombinant expression vector caninclude one or more marker genes, which allow for selection oftransformed or transfected hosts. Marker genes include biocideresistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike. Suitable marker genes for the inventive expression vectorsinclude, for instance, neomycin/G418 resistance genes, hygromycinresistance genes, histidinol resistance genes, tetracycline resistancegenes, and ampicillin resistance genes. The recombinant expressionvector can comprise a native or normative promoter operably linked tothe nucleotide sequence encoding the PCYOX1 inhibitor (includingfunctional portions and functional variants thereof), or to thenucleotide sequence which is complementary to or which hybridizes to thenucleotide sequence encoding the RNA. The selection of promoters, e.g.,strong, weak, inducible, tissue-specific and developmental-specific, iswithin the ordinary skill of the artisan. Similarly, the combining of anucleotide sequence with a promoter is also within the skill of theartisan. The promoter can be a non-viral promoter or a viral promoter,e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSVpromoter and a promoter found in the long-terminal repeat of the murinestem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

In the above IGFBP3 compositions further materials as well as processingtechniques and the like may be set out in Part 5 of Remington'sPharmaceutical Sciences, 20th Edition, 2000, Merck Publishing Company,Easton, Pa., which is incorporated herein by reference.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can also befound in the incorporated materials in Remington's PharmaceuticalSciences. Furthermore, pharmaceutical formulations can be prepared usinga process, which is generally known in the pharmaceutical art.

In the present invention, when the molecule of the invention isadministered with another therapeutic agent, it may be administeredsimultaneously or sequentially.

Sequences

Amino Acid Sequence of IGFBP3:

(SEQ ID No. 4) MQRARPTLWAAALTLLVLLRGPPVARAGASSAGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVSRLRAYLLPAPPAPGEPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK

Nucleotide Sequence of IGFBP3:

Homo sapiens insulin-like growth factor binding protein 3 (IGFBP3),RefSeqGene on chromosome 7, NCBI Reference Sequence: NG_011508.1

mRNA Sequence of IGFBP3:

Homo sapiens insulin-like growth factor binding protein 3 (IGFBP3),transcript variant 1, mRNA, NCBI Reference Sequence: NM_001013398.1

Amino Acid Sequence of TMEM219:

(SEQ ID No. 2) MGNCQAGHNLHLCLAHHPPLVCATLILLLLGLSGLGLGSFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSRLLVLGSFLLLFCGLLCCVTAMCFHPRRESHWSRTRL.

Nucleotide Sequence of TMEM219:

TMEM219 transmembrane protein 219 [Homo sapiens (human)], Gene ID:124446.

mRNA Sequence of TMEM219:

Homo sapiens transmembrane protein 219 (TMEM219), transcript variant 1,mRNA, NCBI Reference Sequence: NM_001083613.1

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will be illustrated by means of non limitingexamples referring to the following figures.

FIGS. 1A-1R. Diabetic enteropathy in long-standing T1D is characterizedby intestinal mucosa abnormalities and impairment in the colonic stemcells. FIGS. 1A-1C are bar graphs depict the score of diarrhea,abdominal pain and constipation according to the administration of theGSRS questionnaire in healthy subjects (CTRL) and long-standing T1Dindividuals (T1D+ESRD). Gray area indicates normal range for theparameter. FIGS. 1D-1F are bar graphs report the measurements ofanorectal sphincter contracting tone (mmHg), reflex response (ml) andurgency volume (ml) by anorectal manometry in healthy subjects (CTRL)and long-standing T1D individuals (T1D+ESRD). Gray area indicates normalrange for the parameter. N=20 CTRL and n=60 T1D+ESRD individuals wereincluded in the evaluation. FIG. 1G, panels G1-G2; FIG. 1I, panelsI1-I2; FIG. 1K, panels K1-K2 FIG. 1M, panels M1-M2; FIG. 1O, panelsO1-O2; FIG. 1O, panels Q1-Q2 are representative images of hematoxylinand eosin (H&E) histology staining, immunostained MIB1⁺ cells,ultrastructural analysis of neural structures indicating localizationand presence of neuroendocrine vesicles, immunostained 5HT⁺, aldehydedehydrogenase (Aldh)⁺ cells, and EphB2⁺ expression, on bioptic samplesobtained from healthy subjects (CTRL) and long-standing T1D individuals(T1D+ESRD). Ultrastructural analysis scale bar: 2000 nm. Originalmagnification: 100× in G1-G2; 400× in I1-I2, K1-K2; 40× in FIG. 1O,panels O1-O2; 200×, in FIG. 1Q, panels Q1-Q2. Scale bar 80 micron. FIGS.1H, 1J, 1L, 1N, 1P, 1R are bar graphs reporting the measurement ofcrypts, MIB1⁺ cells, of neuroendocrine vesicles of nerve terminals(number of cases with >3 NE vesicles detected per nerve terminal), of5HT⁺, Aldh⁺ cells, and of EphB2⁺ expression (intensity score 0-5) inCTRL and long-standing T1D subjects (T1D+ESRD). N=20 CTRL and n=60T1D+ESRD individuals were included in the evaluation. Data are expressedas mean±standard error of the mean (SEM) unless differently reported.*p<0.01; **p<0.001; ***p<0.0001. Abbreviations: GSRS, GastrointestinalSymptom Rating Scale; CoSC, intestinal stem cell; T1D, type 1 diabetes;ESRD, end stage renal disease; CTRL, healthy subjects; H&E, hematoxylinand eosin; MIB1, antibody against Ki67; EphB2, Ephrin B receptor 2;Aldh, Aldehyde dehydrogenase; 5HT, serotonin; NE, neuroendocrinevesicles.

FIGS. 2A-2N. Diabetic enteropathy in long-standing T1D is associatedwith a defect in CoSCs. FIGS. 2A-2B are flow dot plots of EphB2^(low),EphB2^(medium) and EphB2^(hi) cells in healthy subjects (CTRL) andlong-standing T1D individuals (T1D+ESRD). FIGS. 2C-2E are bar graphsdepicting results of flow cytometric analysis of EphB2^(hi+),EphB2^(hi+)LGR5⁺ and EphB2⁺h-TERT⁺ cells in freshly isolated crypts(n=10 CTRL and n=10 T1D+ESRD). FIGS. 2F-2H are bar graphs depictingexpression data of CoSC markers EphB2, LGR5, h-TERT as normalized mRNAexpression measured by quantitative RT-PCR on isolated intestinalcrypts. All samples were run in triplicate and normalized to expressionof the housekeeping gene ACTB (ΔΔCt). FIG. 2I is a scatter plotrepresenting the CoSC signature markers and stem cell transcriptomeprofiling examined in freshly isolated intestinal crypts of n=10 healthysubjects (CTRL) and n=10 long-standing T1D individuals (T1D+ESRD). FIG.2J, panels J1-J2 are representative images of mini-guts cultured for 8days in vitro obtained from previously isolated crypts of long-standingT1D individuals (T1D+ESRD) and healthy subjects (CTRL). 10×magnification. Scale bar 50 micron. FIG. 2K is a bar graph depicting the% of developed mini-guts of the total at 8 days of culture of freshlyisolated intestinal crypts from n=10 CTRL and n=10 T1D+ESRD individuals.FIG. 2L, panels L1-L4 are representative images of mini-guts obtainedfrom previously isolated crypts of healthy subjects (CTRL) and culturedfor 8 days in the following conditions: L1=normal (FBS) serum+normalglucose (5 mM); L2=T1D+ESRD serum+normal glucose; L3=normal serum+highglucose (35 mM); L4=T1D+ESRD serum+high glucose. 10× magnification.Scale bar 50 micron. FIG. 2M is a bar graph depicting the grouping % ofdeveloped mini-guts of the total at 8 days of culture from freshlyisolated intestinal crypts cultured with the following conditions:normal (FBS) serum+normal glucose (5 mM); T1D+ESRD serum+normal glucose;normal serum+high glucose (35 mM); T1D+ESRD serum+high glucose.Statistical significance has been calculated within each group (normalglucose+normal serum, medium+high glucose, medium+long-standing T1Dserum, high glucose+long-standing T1D serum) by comparing differentculturing conditions. Comparison in the bar graph refers to allconditions vs. normal serum+normal glucose. FIG. 2N is a transcriptomeprofile depicting CoSC signature markers expression in isolated cryptsobtained from healthy subjects and cultured with/without high glucoseand/or long-standing T1D serum. N=10 subjects per group were evaluated.Data are expressed as mean±standard error of the mean (SEM) unlessdifferently reported. *p<0.01; **p<0.001; ***p<0.0001. Abbreviations:CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage renaldisease; CTRL, healthy subjects; EphB2, Ephrin B receptor 2; LGR5,leucine-rich repeat containing G protein-coupled receptor 5; RT-PCR,real-time polymerase chain reaction; ACTB, beta actin; FBS, fetal bovineserum.

FIGS. 3A-3M. Circulating IGF-I and IGFBP3 are altered in long-standingT1D and its manipulation in vitro induces profound effects on CoSCgrowth and self-renewal. FIG. 3A is a heat map representing theproteomic profile in long-standing T1D (T1D+ESRD) as compared to healthysubjects (CTRL). The complete dataset of identified and quantifiedproteins was subjected to statistical analysis (p<0.01). Significantlydifferentially expressed proteins were further analyzed throughhierarchical clustering. Sera of n=10 CTRL and n=10 T1D+ESRD individualswere analyzed. FIG. 3B is a bar graph depicting LFQ intensity for asingle protein extrapolated from the untargeted proteomic analysis,insulin-like growth factor binding protein 3 (IGFBP3). FIG. 3C, panelsC1-C2 are representative images (40× magnification) of IGFBP3 expressionin the liver. IGFBP3 is mildly and diffusely expressed in the liverparenchyma from healthy subjects (FIG. 3C, panel C1), while it is morezonally positive in long-standing diabetic individuals (FIG. 3C, panelC2). FIG. 3D is a bar graph representing IGFBP3 levels measured by ELISAin the supernatants of immortalized human hepatoma cell line (HuH-7)cultured for 5 days at different glucose concentrations (35 mM: highglucose; 20 mM: intermediate glucose; 5 mM: normal glucose). Experimentswere run in triplicate. FIG. 3E is a bar graph representing insulin-likegrowth factor 1 (IGF-I) levels measured by ELISA in serum of healthysubjects and long-standing T1D (T1D+ESRD). FIG. 3F is a Western blotanalysis (cropped blots) confirmed IGF-IR and TMEM219 expression on theintestinal crypt surface. Evaluation of total IGF-IR expression by WBincludes the detection of IGF-IRa, a subunit of IGF-IR whole protein.Representative pictures of TMEM219 in situ hybridization (FIG. 3G, panelG1 negative control; FIG. 3G, panel G2 TMEM219 staining) performed onrectal mucosa biopsy samples obtained from CTRL. 20× magnification. FIG.3G, panels G1-G2 are representative pictures of TMEM219 in situhybridization (FIG. 3G, panrl G1 negative control, FIG. 3G, panel G2TMEM219 staining) performed on rectal mucosa biopsy samples obtainedfrom CTRL. Magnification 400×. FIG. 3H is a bar graph depictingnormalized mRNA expression of TMEM219 (IGFBP3 receptor) using the ΔΔCtmethod. N=5 subjects per group were evaluated. FIG. 3I is a bar graphdepicting the grouping % of developed mini-guts of the total obtainedfrom long-standing T1D individuals in different conditions and showingthe effect of IGF-I, IGFBP3 and anti-IGF-IR. The p values are relativeto baseline conditions and addition of IGF-I to culture. FIG. 3J is abar graph representing normalized mRNA expression of Caspase 8 and 9 incrypts isolated from healthy subjects cultured in the presence of IGFBP3and IGF-I+IGFBP3, performed in triplicate. FIG. 3K is a bar graphdepicting the grouping % of developed mini-guts of the total at 8 daysof culture, obtained from healthy subjects and cultured in the presenceof a Pan-Caspase inhibitor, selective inhibitors of Caspase 8, 9 and 3,and IGFBP3. Assay was performed in triplicate. FIG. 3L are bar graphsdepicting the grouping % of developed mini-guts of the total obtainedfrom healthy subjects and cultured in different conditions (normalglucose+normal serum, high glucose+normal serum, T1D+ESRD serum+normalglucose, T1D+ESRD serum+high glucose) and showing the effect of IGF-I,IGFBP3 and anti-IGF-IR. The p values are relative to baseline condition(medium alone, medium+high glucose, medium+long-standing T1D serum, highglucose+long-standing T1D serum). Additional p values have beencalculated to compare the difference in mini-gut growth among thefollowing conditions: medium alone vs. medium+high glucose, vs.medium+high glucose+long-standing T1D serum). Assay was performed intriplicate. FIG. 3M is a bar graph depicting the grouping % of developedmini-guts of the total obtained from healthy subjects, cultured for 8days, exposed to TMEM219 targeting with siRNA and finally compared toTMEM219-expressing crypts in medium alone and in medium+highglucose+long-standing T1D serum. Assay was performed in triplicate. Dataare expressed as mean±standard error of the mean (SEM) unlessdifferently reported. *p<0.01; **p<0.001; ***p<0.0001. Abbreviations:IGF-I, insulin-like growth factor 1; IGFBP3, insulin-like growth factorbinding protein 3; IGF-IR, insulin-like growth factor 1 receptor; CoSC,colonic stem cell; T1D, type 1 diabetes; ESRD, end stage renal disease;CTRL, healthy subjects; RT-PCR, real-time polymerase chain reaction;ACTB, beta actin; LFQ, Label-free quantitation; SEM, standard error ofthe mean; siRNA, small RNA interference; inhib, inhibitor.

FIGS. 4A-4Q. Effects of the peripheral IGF-I/IGFBP3 dyad on single-cellderived in vitro mini-guts and on caspase cascade. Manipulating theperipheral IGF-I/IGFBP3 dyad alters the progression of diabeticenteropathy in a preclinical model of diabetic enteropathy, while thetreatment of long-standing T1D with simultaneous pancreas-kidneytransplantation (SPK) ameliorates intestinal symptoms, motility andmorphology. FIG. 4A is a bar graph representing normalized mRNAexpression of TMEM219, LRP1, TGF-β type I and II, in EphB2⁺ sortedsingle cells obtained from crypts of healthy subjects. Experiments wereperformed in triplicate. FIG. 4B is a bar graph showing % of developedsingle cell-derived mini-guts (of the total) obtained from EphB2⁺ cellssorted from freshly isolated crypts of healthy subjects and cultured indifferent conditions (normal glucose+normal serum, high glucose+normalserum, T1D+ESRD serum+normal glucose, T1D+ESRD serum+high glucose) andshowing the effect of IGF-I and IGFBP3. The p values are relative tobaseline condition. FIGS. 4C-4D are scatter plots representing theapoptosis transcriptome profiling examined in freshly isolatedintestinal crypts of healthy subjects (CTRL) and long-standing T1Dindividuals (T1D+ESRD) cultured with/without IGFBP3 and IGF-I.Experiments were run in triplicate. FIG. 4E is a schematic attempt torepresent the effect of circulating IGF-I and IGFBP3 on the CoSCs. FIGS.4F, 4G, 4I are line graphs reporting the number of crypts (FIG. 4F),depth of crypts (FIG. 4G) and width of crypts (FIG. 4I) assessed onintestinal lower tract sections harvested at baseline and after 8 weeksfrom STZ-treated B6 mice developing diabetic enteropathy (B6+STZ), näiveB6 (WT), and näive B6 treated with IGFBP3 (WT+IGFBP3). WT: wild type,STZ: streptozoticin-treated. N=3 mice per group were evaluated. FIG. 4H,panels H1-H3 are representative images of intestinal crypts on H&Esections of WT, B6+STZ mice developing diabetic enteropathy, and näiveB6 treated with IGFBP3 (WT+IGFBP3). Histology magnification, 400×. FIG.4J is a bar graph representing the number of Aldh⁺ cells/mm² inimmunostained sections of STZ-treated B6 mice developing diabeticenteropathy, WT, and näive B6 treated with IGFBP3 (WT+IGFBP3). FIG. 4K,panels K1-K3 are representative images of Aldh⁺ cells on immunostainedsections of intestinal lower tract harvested from STZ-treated B6 micedeveloping diabetic enteropathy, WT, and näive B6 treated with IGFBP3(WT+IGFBP3). Histology magnification, 400×. FIGS. 4L, 4N, 4P are bargraphs reporting the measurement of MIB1⁺ and Aldh⁺ cells, and EphB2⁺expression (intensity score 0-5) in the four groups of subjects (n=20CTRL, n=30 SPK, n=K+T1D and n=60 T1D+ESRD). FIG. 4M, panels M1-M21; FIG.4O, panels O1-O2; FIG. 4Q, panels Q1-Q2 are representative images ofMIB1⁺ and Aldh⁺ cells, and EphB2⁺ expression in immunostained rectalmucosa bioptic samples of T1D+ESRD who underwent kidney alone (K+T1D) orsimultaneous pancreas-kidney (SPK) transplantation at 8 years offollow-up. Histology 400× in M1-M2 and 01-02, 20× in Q1-Q2. Scale bar 80micron. Data are expressed as mean±standard error of the mean (SEM)unless differently reported. *p<0.01; **p<0.001; ***p<0.0001.Abbreviations: WT, wild type; STZ, streptozoticin-treated; B6, C57BL/6Jmice; IGF-I, insulin-like growth factor 1; IGFBP3, insulin-like growthfactor binding protein 3; IGF-IR, insulin-like growth factor 1 receptor;CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage renaldisease; CTRL, healthy subjects; SPK, simultaneous kidney-pancreastransplantation; K+T1D, kidney transplantation alone in type 1 diabetes;H&E, hematoxylin and eosin; MIB1, antibody against Ki67; EphB2, Ephrin Breceptor 2; Aldh, Aldehyde dehydrogenase; SEM, standard error of themean.

FIGS. 5A-5M. Treatment of long-standing T1D with SPK replenishes CoSCsand restores the CoSC signature profile and mini-gut development throughrestoration of circulating IGF-I and IGFBP3. FIGS. 5A-5C are bar graphsdepicting results of flow cytometric analysis of EphB2^(hi+),EphB2^(hi+)LGR5⁺, EphB2⁺h-TERT⁺ cells obtained from isolated crypts inlong-standing T1D (Baseline), T1D+ESRD who underwent kidney pancreas(SPK) or kidney alone (K+T1D) transplantation at 8 years of follow-up.N=10 subjects per group were evaluated. FIGS. 5D-5F are bar graphsdepicting normalized mRNA expression of intestinal stem cell markersEphB2, LGR5, h-TERT, measured by quantitative RT-PCR on isolatedintestinal crypts obtained from long-standing T1D (Baseline), T1D+ESRDwho underwent kidney pancreas (SPK) or kidney alone (K+T1D)transplantation at 8 years of follow-up. All samples were run intriplicate and normalized to expression of the housekeeping gene ACTBusing the ΔΔCt method. N=10 subjects per group were evaluated. FIG. 5Gis a Western blot analysis depicting the expression of EphB2, LGR5,h-TERT in isolated intestinal crypts of the four groups at 8 years offollow-up. N=5 subjects per group were evaluated. FIG. 5H is a bar graphdepicting the % of developed mini-guts of the total at 8 days of cultureof freshly isolated intestinal crypts obtained from long-standing T1Dindividuals (Baseline), SPK and K+T1D subjects at 8 years of follow-up.N=10 subjects per group were evaluated. FIG. 5I is a heat maprepresenting the CoSC signature marker transcriptomic profiling examinedin freshly isolated intestinal crypts of CTRL, long-standing T1Dindividuals (T1D+ESRD), SPK and K+T1D subjects at 8 years of follow-up.N=10 subjects per group were evaluated. FIG. 5J is a bar graphrepresenting IGF-I levels measured by ELISA in serum of the four groupsof subjects at 8 years of follow-up. N=10 subjects per group wereevaluated. FIG. 5K is a bar graph depicting IGFBP3 levels measured byELISA in serum of the four groups of subjects. N=20 subjects per groupwere evaluated. FIGS. 5L-5M are scatter plots depicting the correlationbetween IGFBP3 serum levels and intestinal symptoms assessed using theGSRS questionnaire (0-7) in n=20 subjects of K+T1D (L) and SPK (M)group. Analysis was conducted using ANOVA (p<0.05) in comparing allgroups. Data are expressed as mean±standard error of the mean (SEM)unless differently reported. *p<0.01; **p<0.001; ***p<0.0001.Abbreviations: CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, endstage renal disease; CTRL, healthy subjects; SPK, simultaneouskidney-pancreas transplantation; EphB2, Ephrin B receptor 2; LGR5,leucine-rich repeat containing G protein-coupled receptor 5; RT-PCR,real-time polymerase chain reaction; ACTB, beta actin; K+T1D, kidneytransplantation alone in type 1 diabetes; IGF-I, insulin-like growthfactor 1; IGFBP3, insulin-like growth factor binding protein 3; SEM,standard error of the mean.

FIGS. 6A-6N. Treatment with the newly generated recombinant proteinecto-TMEM219 (ecto-TMEM219) abrogates IGFBP3-mediated mini-gutdestruction and preserves CoSCs in preclinical model. FIG. 6A is a bargraph depicting the grouping % of developed mini-guts of the totalobtained from healthy subjects in different conditions and showing theeffect of ecto-TMEM219 at various concentrations (1:2, 1:1 and 2:1 molarratio as compared to IGFBP3) in IGFBP3-treated mini-guts and in thoseexposed to high glucose. The p values are relative to baselineconditions. FIG. 6B is a bar graph representing normalized mRNAexpression of EphB2 in crypts isolated from healthy subjects cultured inthe presence of IGFBP3 and ecto-TMEM219+IGFBP3, performed in triplicate.FIGS. 6C-6D are bar graphs representing normalized mRNA expression ofCaspase 8 and 9 in crypts isolated from healthy subjects cultured in thepresence of IGFBP3 and ecto-TMEM219+IGFBP3, performed in triplicate.FIGS. 6E-6G are bar graphs reporting the number of crypts (FIG. 6E),depth of crypts (FIG. 6F) and width of crypts (FIG. 6G) assessed onintestinal lower tract sections harvested at baseline and after 8 weeksfrom STZ-treated B6 mice developing diabetic enteropathy (B6+STZ), naïveB6 (WT), and STZ-B6 mice treated with ecto-TMEM219. WT: wild type, STZ:streptozoticin-treated. N=3 mice per group were evaluated. FIG. 6H is aline graph reporting the weight at baseline and after 8 weeks ofSTZ-treated B6 mice developing diabetic enteropathy (B6+STZ), naïve B6(WT), and of STZ-treated B6 mice developing diabetic enteropathy treatedwith ecto-TMEM219. WT: wild type, STZ: streptozoticin-treated. N=3 miceper group were evaluated. FIG. 6I is a bar graph representing results offlow cytometric analysis of EphB2⁺ cells isolated from intestinalsamples collected from naïve B6 mice, STZ-treated B6 mice and in STZ-B6mice treated with ecto-TMEM219 at 8 weeks. FIG. 6J consists ofrepresentative flow histograms of EphB2⁺ cells isolated from cryptsisolated from naïve B6 mice, STZ-treated B6 mice and in STZ-B6 micetreated with ecto-TMEM219 at 8 weeks. N=3 to 5 mice per group wereevaluated. FIG. 6K is a bar graph representing normalized mRNAexpression of EphB2 in intestinal samples collected from naïve B6 mice,STZ-treated B6 mice and in STZ-B6 mice treated with ecto-TMEM219 at 8weeks. FIGS. 6L-6M are bar graphs representing normalized mRNAexpression of Caspase 8 (K) and Caspase 9 (L) in intestinal samplescollected from naïve B6 mice, STZ-treated B6 mice and in STZ-B6 micetreated with ecto-TMEM219 at 8 weeks. FIG. 6N is a bar graphrepresenting IGFBP3 circulating levels measured in naïve B6 mice (WT)and STZ-treated B6 mice (B6+STZ) and in B6+STZ mice treated withecto-TMEM219 at 8 weeks. Data are expressed as mean±standard error ofthe mean (SEM) unless differently reported. *p<0.01; **p<0.001;***p<0.0001. Abbreviations: WT, wild type; STZ, streptozoticin-treated;B6, C57BL/6J mice; IGF-I, insulin-like growth factor 1; IGFBP3,insulin-like growth factor binding protein 3; CoSC, colonic stem cell;H&E, hematoxylin and eosin; EphB2, Ephrin B receptor 2; SEM, standarderror of the mean, T1D, type 1 diabetes; ESRD, end stage renal disease;CTRL, healthy subjects; RT-PCR, real-time polymerase chain reaction;ACTB, beta actin.

FIGS. 7A-7F. Assessment of IGFBP3 levels in serum (FIG. 7A) and urine(FIG. 7B) of CTRL, T1D and T1D+ESRD individuals shown in bar graphs.(FIG. 7C) A scatter plot showing the correlation between serum and urineIGFBP3 levels in all subjects of the cohort evaluated for this study.FIGS. 7D-7E are scatter plots showing the correlation between IGFBP3serum levels and eGFR calculated with MDRD formula in subjects withT1D+ESRD on dialysis (FIG. 7D) and with T1D with eGFR >15 ml/min/m2(FIG. 7E). FIG. 7F is a scatter plot showing the correlation betweenserum and urine IGFBP3 levels in all subjects of the cohort evaluatedfor this study. The gray area indicates the normal range within urinaryand serum levels of IGFBP3.

FIGS. 8A-8N. CoSC profile, in vitro generation of mini-guts, expressionof IGFBP3 in the liver and of IGF-IR on CoSCs in long-standing T1D andhealthy subjects. FIGS. 8A-8B are representative flow dot plots of PI⁻cells gating strategy in healthy subjects (CTRL) and long-standing T1Dindividuals (T1D+ESRD). FIG. 8C is a bar graph depicting the results offlow cytometric analysis of PI⁻ cells in freshly isolated crypts (n=10CTRL and n=10 T1D+ESRD). FIGS. 8D-8E are representative flow dot plotsof EphB2^(hi)LGR5⁺ (D) and EphB2⁺h-TERT⁺ cells in healthy subjects(CTRL) and long-standing T1D individuals (T1D+ESRD). FIG. 8F is aWestern blot analysis (cropped blots) confirms low expression of EphB2,LGR5, h-TERT in in vitro isolated intestinal crypts of long-standing T1Dindividuals (T1D+ESRD). Full-length blots are presented in FIG. 5G. N=5subjects per group were evaluated. FIG. 8G is a scatter plotrepresenting the stem cell transcriptome profiling examined in freshlyisolated intestinal crypts of healthy subjects (CTRL) and long-standingT1D individuals (T1D+ESRD). A table summarizes genes and pathwaysanalyzed (Table S1). N=10 subjects per group were evaluated. FIGS. 8H-8Iare representative images of freshly isolated crypts obtained fromhealthy subjects and long-standing T1D individuals stained with DAPI.20× magnification. FIG. 8J is a bar graph representing the percentage ofmini-guts forming efficiency of plated crypts obtained from healthysubjects and long-standing T1D individuals at 12 hours. N=10 subjectsper group were evaluated. FIG. 8K is a bar graph representing thecalculated combined score of IGFBP3 intensity/diffusion (0-6) uponimmunohistochemical evaluation in liver samples obtained from healthysubjects and long-standing T1D individuals. N=3 subjects per group wereevaluated. FIG. 8L, panels L1-L6 representative images (63×magnification) of IGFBP3 expression in the liver. Immunofluorescenceconfirmed the colocalization of Hep Par-1⁺ cells and IGFBP3 expression(FIG. 8L, panels L1-L3), while no colocalization was observed betweenIGFBP3 and CD163⁺ cells (FIG. 8L, panels L4-L6). FIG. 8M is a bar graphdepicting normalized mRNA expression of the IGF-I receptor (IGF-IR)measured by quantitative RT-PCR on isolated intestinal crypts. Allsamples were run in triplicate and normalized to the housekeeping geneACTB using the ΔΔCt method. FIG. 8N, panels N1-N2 are representativepictures of IGF-IR⁺ cells on rectal mucosa samples obtained from CTRLand from T1D+ESRD individuals. Black arrow indicates positive cells atthe crypt base. Magnification 200×. Data are expressed as mean±standarderror of the mean (SEM) unless differently reported. *p<0.01.Abbreviations: PI, propidium iodide; IGF-I, insulin-like growth factor1; IGFBP3, insulin-like growth factor binding protein 3; IGF-IR,insulin-like growth factor 1 receptor; CoSC, colonic stem cell; T1D,type 1 diabetes; ESRD, end stage renal disease; CTRL, healthy subjects;EphB2, Ephrin B receptor 2; LGR5, leucine-rich repeat containing Gprotein-coupled receptor 5; RT-PCR, real-time polymerase chain reaction;ACTB, beta actin; SEM, standard error of the mean.

FIGS. 9A-9H. Caspases expression in IGF-I/IGFBP3 cultured mini-guts andthe lack of effect of other circulating factors confirmed IGFBP3 majorpro-apoptotic effect on mini-guts development. FIG. 9A is a bar graphrepresenting normalized mRNA expression of Caspase 8 in crypts isolatedfrom individuals with T1D+ESRD cultured in the presence of IGFBP3,IGF-I+IGFBP3 and IGF-I, performed in triplicate. FIG. 9B is a bar graphrepresenting normalized mRNA expression of Caspase 9 in crypts isolatedfrom individuals with T1D+ESRD cultured in the presence of IGFBP3,IGF-I+IGFBP3 and IGF-I, performed in triplicate. FIGS. 9C-9D are bargraphs depicting the grouping % of mini-guts developed from healthysubjects (C) and from long-standing T1D individuals (D), cultured in thepresence of medium with FBS and medium with serum obtained from healthysubjects, “CTRL serum”. Assay was run in triplicate. FIG. 9E is a bargraph grouping % of developed mini-guts of the total obtained fromhealthy subjects, cultured for 8 days, exposed to TMEM219 targeting withsiRNA and anti-IGF-IR, and finally compared to TMEM219-expressing cryptsin medium alone and in medium+high glucose+long-standing T1D serum.Assay was performed in triplicate. FIGS. 9F-9G are bar graphsillustrating the grouping % of developed mini-guts at 8 days of culture,obtained from healthy subjects (FIG. 9F) and long-standing T1Dindividuals (FIG. 9G) cultured in the presence of medium alone andvarious molecules identified with proteomic analysis (Table S7). Assaywas performed in triplicate. FIG. 9H is a bar graph depicting thegrouping % of mini-guts obtained from healthy subjects and cultured inthe presence of medium alone, medium+high glucose, medium+high glucoseand long-standing T1D serum, IGF-I, IGFBP3 with/without insulin. Assaywas performed in triplicate. Data are expressed as mean±standard errorof the mean (SEM) unless differently reported. *p<0.01; **p<0.001.Abbreviations: IGF-I, insulin-like growth factor 1; IGFBP3, insulin-likegrowth factor binding protein 3; IGF-IR, insulin-like growth factor 1receptor; CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stagerenal disease; CTRL, healthy subjects; RT-PCR, real-time polymerasechain reaction; ACTB, beta actin; SEM, standard error of the mean;siRNA, small RNA interference; ALDOA, Fructose-bisphosphate aldolase A;RNASE, Ribonuclease pancreatic; MASP, Mannan-binding lectin serineprotease 1.

FIGS. 10A-10J. Effect of IGF-I/IGFBP3 dyad on single cell derivedmini-guts, on stem cell transcriptome profile and on apoptotic pathways.FIG. 10A, panels, A1-A3 are representative images of single cell-derivedmini-guts, cultured for 8 days in vitro obtained from previouslyisolated EphB2⁺ sorted cells of healthy subjects and cultured withmedium alone, medium+IGFBP3, medium+Glucose 35 mM+long-standing T1Dserum (T1D+ESRD). Images are shown at 10× magnification. Scale bar 50micron. FIGS. 10B-10D are bar graphs representing normalized mRNAexpression of Caspase 8, Caspase 9 and Ki67 in single cell-derivedmini-guts grown from flow sorted EphB2⁺ cells isolated from healthysubjects and cultured in different conditions. Assay was performed intriplicate. FIGS. 10E-10F are scatter plots representing the stem celltranscriptome profiling examined in freshly isolated intestinal cryptsof healthy subjects (CTRL) and long-standing T1D individuals (T1D+ESRD)cultured with/without IGFBP3 and IGF-I. Assays were run in triplicate.FIGS. 10G-10H are scatter plots representing the apoptosis transcriptomeprofiling examined in freshly isolated intestinal crypts of healthysubjects (CTRL) and long-standing T1D individuals (T1D+ESRD) culturedwith/without IGF-I. A table summarizes genes and pathways analyzed(Table S3). Assays were run in triplicate. FIGS. 10I-10J are bar graphsdepicting the grouping % of mini-guts developed from crypts obtainedfrom healthy subjects (FIG. 10I) and long-standing T1D (T1D+ESRD) (FIG.10J) and then cultured in the presence of medium alone, Fas Ligand(FasL), hydrogen peroxide (H₂O₂) and Tumor Necrosis Factor alpha(TNF-α). Assay was performed in triplicate. Data are expressed asmean±standard error of the mean (SEM) unless differently reported.*p<0.01; **p<0.001; ***p<0.0001. Abbreviations: IGF-I, insulin-likegrowth factor 1; IGFBP3, insulin-like growth factor binding protein 3;CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage renaldisease; CTRL, healthy subjects; RT-PCR, real-time polymerase chainreaction; ACTB, beta actin; SEM, standard error of the mean; FasL, FasLigand; H₂O₂, hydrogen peroxide; TNF-α, Tumor Necrosis Factor alpha.

FIGS. 11A-11N. Manipulating IGF-I/IGFBP3 dyad in preclinical models ofdiabetic enteropathy. FIG. 11A is a bar graph representing IGFPB3circulating levels measured in naïve B6 mice (WT) and STZ-treated B6mice (B6+STZ). FIG. 11B is a bar graph representing IGF-I circulatinglevels measured in naïve B6 mice (WT) and STZ-treated B6 mice (B6+STZ).FIG. 11C is a bar graph representing insulin serum levels measured innaïve B6 mice (WT) and STZ-treated B6 mice (B6+STZ). FIGS. 11D-11F arebar graphs reporting the number of crypts (FIG. 11D), depth of crypts(FIG. 11E) and width of crypts (FIG. 11F) assessed on intestinal lowertract sections harvested at baseline and after 8 weeks from STZ-treatedB6 mice developing diabetic enteropathy (B6+STZ), naïve B6 (WT), andSTZ-B6 mice treated with IGFBP3 (B6+STZ+IGFBP3) or with IGF-I(B6+STZ+IGF-I). WT: wild type, STZ: streptozoticin-treated. N=3 mice pergroup were evaluated. FIG. 11G is a bar graph representing the number ofAldh⁺ cells/mm² in immunostained sections of STZ-treated B6 micedeveloping diabetic enteropathy, WT, and STZ-B6 mice treated with IGFBP3(B6+STZ+IGFBP3) or with IGF-I (B6+STZ+IGF-I). FIG. 11H, panels H1, H2are representative images of intestinal crypts on H&E sections of STZ-B6mice treated with IGFBP3 (B6+STZ+IGFBP3), (FIG. 11H, panel H1) or withIGF-I (B6+STZ+IGF-I), (FIG. 11H, panel H2). Histology magnification,400×. FIG. 11I is a line graph reporting the weight of STZ-treated B6mice developing diabetic enteropathy (B6+STZ), naïve B6 (WT),STZ-treated B6 mice developing diabetic enteropathy treated with IGFBP3(B6+STZ+IGFBP3). WT: wild type, STZ: streptozoticin-treated. N=3 miceper group were evaluated. FIG. 11J is a bar graph representing resultsof flow cytometric analysis of EphB2⁺ cells in intestinal samplescollected from naïve B6 mice, STZ-treated B6 mice and in STZ-B6 micetreated with IGFBP3 (B6+STZ+IGFBP3). FIGS. 11K-11L are bar graphsrepresenting normalized mRNA expression of EphB2 (FIG. 11K) and LGR5(FIG. 11L) in intestinal samples collected from naïve B6 mice,STZ-treated B6 mice and in STZ-B6 mice treated with IGFBP3(B6+STZ+IGFBP3). FIGS. 11M-11N are bar graphs representing normalizedmRNA expression of Caspase 8 (FIG. 11M) and Caspase 9 (FIG. 11N) inintestinal samples collected from naïve B6 mice, STZ-treated B6 mice andin STZ-B6 mice treated with IGFBP3 (B6+STZ+IGFBP3). Data are expressedas mean±standard error of the mean (SEM) unless differently reported.*p<0.01; **p<0.001; ***p<0.0001. Abbreviations: WT, wild type; STZ,streptozoticin-treated; B6, C57BL/6J mice; IGF-I, insulin-like growthfactor 1; IGFBP3, insulin-like growth factor binding protein 3; CoSC,colonic stem cell; H&E, hematoxylin and eosin; EphB2, Ephrin B receptor2; Aldh, Aldehyde dehydrogenase; SEM, standard error of the mean.

FIGS. 12A-12K. The treatment of long-standing T1D with SPK amelioratesdiabetic enteropathy. FIGS. 12A-12C are bar graphs depicting the scoreof abdominal pain, diarrhea and constipation according to the GSRSquestionnaire in healthy subjects (CTRL), long-standing T1D individuals(Baseline), T1D+ESRD who underwent kidney pancreas (SPK) or kidney alone(K+T1D) transplantation. Gray area indicates normal range for all theparameters. Statistics are expressed as mean±SEM. FIG. 12D, panelsD1-D2; FIG. 12E, panels E1-E2; FIG. 12G, panels G1-G2; FIG. 12J, panelsJ1-J2 are representative pictures of hematoxylin and eosin (H&E) (FIG.12D) staining and ultrastructural analysis of neural structures indicatelocalization and presence of neuroendocrine vesicles (FIG. 12E),staining and ultrastructural analysis of Schwann cells indicatecytoplasm derangements (FIG. 12G), and 5HT⁺ cells (12J) performed onrectal mucosa biopsy samples obtained from T1D+ESRD who underwent kidneypancreas (SPK) or kidney alone (K+T1D) transplantation at 8 years offollow-up. Magnification 400×. FIG. 12F, FIG. 12H, FIG. 12I, FIG. 12Kare bar graphs reporting the measurements of neuroendocrine vesicles (%of cases with >3 NE vesicles detected per nerve terminal), % of Schwanncells with picnotic nuclei and cytoplasm derangements (% of positivecases) using electron microscopy, 5HT⁺ cells, performed on biopticsamples obtained from rectal mucosa of CTRL, long-standing T1Dindividuals (Baseline), T1D+ESRD who underwent kidney pancreas (SPK) orkidney alone (K+T1D) over an 8-year follow-up period. Statistics areexpressed as mean±SEM. N=20 CTRL, n=30 SPK, n=30 K+T1D and n=60 T1D+ESRDsubjects were evaluated. Statistics are expressed as mean±SEM. Allparameters examined were statistically significantly different whencomparing different groups as following: *p<0.01; **p<0.001;***p<0.0001. N=10 subjects per group were evaluated. Abbreviations:GSRS, Gastrointestinal Symptom Rating Scale; SPK, simultaneouskidney-pancreas transplantation; K+T1D, kidney transplantation alone intype 1 diabetes; CTRL, healthy subjects; T1D, type 1 diabetes; ESRD, endstage renal disease; 5HT, serotonin; H&E, hematoxylin and eosin; NGF,neural growth factor; SEM, standard error of the mean; NE,neuroendocrine vesicles.

FIGS. 13A-13D. Analysis of colonic stem cells, IGF-IR and proteomicprofile of circulating factors in diabetic enteropathy in SPK and K+T1Dgroups. FIG. 13A, panels A1-A6 are representative images of mini-guts,cultured for 8 days in vitro obtained from previously isolated crypts oflong-standing T1D individuals, T1D+ESRD who underwent kidney pancreas(SPK) or kidney alone (K+T1D) transplantation at 8 years of follow-up.Images are shown at 5× and 10× magnification. Scale bar 10 micron. FIG.13B is a scatter plot representing the stem cell transcriptome profilingexamined in freshly isolated intestinal crypts of SPK individuals. N=3subjects were evaluated. FIG. 13C is a bar graph depicting relativeexpression levels of IGF-I receptor (IGF-IR) on isolated crypts ofhealthy subjects (CTRL), long-standing T1D individuals (T1D+ESRD), SPKand K+T1D measured by quantitative RT-PCR. All samples were run intriplicate and normalized to the ACTB relative expression level usingthe ΔΔCt method. Results are expressed as mean±SEM. FIG. 13D is a heatmap that represents the proteomic profile of long-standing T1D ascompared to CTRL and SPK subjects at 8 years of follow-up. The completedataset of identified and quantified proteins was subjected tostatistical analysis (p<0.05). Significantly differentially expressedproteins were further analyzed through hierarchical clustering.Statistics are expressed as mean±SEM. Sera of n=10 subjects per groupwere evaluated. All parameters examined were statistically significantlydifferent when comparing different groups as following: *p<0.01.Abbreviations: T1D, type 1 diabetes; ESRD, end stage renal disease;CTRL, healthy subjects; SPK, simultaneous kidney-pancreastransplantation; K+T1D, kidney transplantation alone in type 1 diabetes;RT-PCR, real-time polymerase chain reaction; ACTB, beta actin; IGF-I,insulin-like growth factor 1; IGFBP3, insulin-like growth factor bindingprotein 3; IGF-IR, insulin-like growth factor 1 receptor; SEM, standarderror of mean.

FIGS. 14A-14G. Correlation of intestinal symptoms with levels ofinsulin, HbA1C and blood glucose in SPK and K+T1D groups. FIGS. 14A-14Bare scatter plots showing a correlation between insulin serum levels andintestinal symptoms assessed using the GSRS questionnaire andconsidering the item with the highest score (0-7) in n=20 subjects ofK+T1D (FIG. 14A) and SPK (FIG. 14B) group. Analysis was conducted usingANOVA (p<0.05) in comparing all groups. FIG. 14C is a bar graphdepicting insulin serum levels measured using the Free-insulin method inn=20 subjects of K+T1D (FIG. 14A) and SPK (FIG. 14B) group. Data areexpressed as mean±standard error of the mean (SEM). FIGS. 14D-14E arescatter plots showing the correlation between glycated hemoglobin(HbA1C) serum levels and intestinal symptoms assessed using the GSRSquestionnaire (0-7) in n=20 subjects of K+T1D (FIG. 14A) and SPK (FIG.14B) group. Analysis was conducted using ANOVA (p<0.05) in comparing allgroups. FIGS. 14F-14G are scatter plots showing the correlation betweenblood glucose levels (Glycemia) and intestinal symptoms assessed usingthe GSRS questionnaire (0-7) in n=20 subjects of K+T1D (FIG. 14A) andSPK (FIG. 14B) group. Analysis was conducted using ANOVA (p<0.05) incomparing all groups. Abbreviations: T1D, type 1 diabetes; ESRD, endstage renal disease; CTRL, healthy subjects; SPK, simultaneouskidney-pancreas transplantation; K+T1D, kidney transplantation alone intype 1 diabetes; IGF-I, insulin-like growth factor 1; IGFBP3,insulin-like growth factor binding protein 3.

FIGS. 15A-15F. Expression of cell lineages markers in mini-guts exposedto different culturing conditions. FIG. 15A, panels A1-A4; FIG. 15B,panels B1-B4; FIG. 15C, panels C1-C4; FIG. 15D, panels D1-D4; FIG. 15E,panels E1-E4 are representative images (10× magnification) ofcitokeratin 20 (KRT20), vimentin, Synaptofisin and AldehydeDehydrogenase (ALDH) expression in mini-guts obtained from cryptsisolated from healthy subjects, CTRL (FIG. 15A, panels A1-A4), andT1D+ESRD individuals (FIG. 15B, panels B1-B4), cultured with IGFBP3(FIG. 15C, panels C1-C4), Glucose 35 mM (FIG. 15D, panels D1-D4), andGlucose 35 mM)+long-standing T1D serum (T1D+ESRD serum)+IGF-I (FIG. 15E,panels E1-E4). Immunofluorescence confirmed that expression of alllineages markers is reduced in mini-guts obtained from T1D+ESRDindividuals as compared to CTRL (FIG. 15A, panels A1-A4; FIG. 15B,panels B1-B4), with ALDH being the least expressed marker (FIG. 15B,panel B4). Decreased ALDH expression was also detected in IGFBP3-treatedmini-guts (FIG. 15C, panel C4), while mini-guts exposed to high glucoseand long-standing T1D serum and treated with IGF-I showed evident ALDHexpression recovery. FIG. 15F is a bar graph representing expression ofTMEM219, KRT20, Epithelial-cell adhesion molecule (EpCam) andChromogranin A (CHGA) on non-stem cells (EphB2⁻ cells) measured byquantitative RT-PCR. All samples were run in triplicate and normalizedto the ACTB relative expression level using the ΔΔCt method. Results areexpressed as mean±SEM. Abbreviations: T1D, type 1 diabetes; ESRD, endstage renal disease; CTRL, healthy subjects; IGF-I, insulin-like growthfactor 1; IGFBP3, insulin-like growth factor binding protein 3; IF,immunofluorescence; KRT20, citokeratin 20, ALDH, Aldehyde Dehydrogenase,EpCam, epithelial cell adhesion molecule; CHGA, Chromogranin A; RT-PCR,real-time polymerase chain reaction; ACTB, beta actin.

FIG. 16. Selection strategy to test candidate proteins in in vitromini-guts assay. FIG. 16 is a flow chart depicting the strategy used toselect protein candidates based on proteomic profile to be tested invitro mini-guts assay.

FIGS. 17A-17G; 17M-17P. Analysis of developed mini-guts using the cryptdomain quantitative criteria. FIGS. 17A-17G; 17M-17P are bar graphsgrouping % of developed mini-guts with at least 1 crypt domaindetectable in different conditions already reported throughout thepaper.

FIG. 18 consists of three bar graphs illustrating the peripheral IGFBP3levels are increased in individuals with inflammatory bowel disease ascompared to healthy subjects.

FIGS. 19A-19B. IGFBP3 peripheral levels are increased in pre-diabeticand diabetic conditions in T1D (FIG. 19A) and T2D human subjects (FIG.19B). *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: IGFBP3,insulin-like growth factor binding protein 3; CTRL, healthy subjects;T1D, type 1 diabetes; T2D, type 2 diabetes; AutoAb positive: nondiabetic subjects at risk for developing T1D with detected positivity ofAntibodies against islets peptides; IGT: impaired glucose tolerancemeasured at the OGTT (oral glucose tolerance test) in fasting andnon-fasting condition. NGT: normal glucose tolerance measured at theOGTT. IFG: impaired fasting glucose tolerance measured at OGTT andresulting positive only in fasting conditions.

FIGS. 20A-20B. IGFBP3 peripheral levels increase in pre-diabetic anddiabetic conditions in murine models of T1D (FIG. 20A) and T2D (FIG.20B). *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: C57BL6/J, B6 mice;B6, naïve mice; NOD, non-obese diabetic mice; HI-D, high-fat diet,IGFBP3, insulin-like growth factor binding protein 3.

FIGS. 21A-21B. IGFBP3 production in primary human hepatocytes increasesduring glucose exposure (11 mM, 20 mM, 35 mM) (FIG. 21A) andinflammation (IFNγ 1,000 U/ml, plus I1-1β 2 ng/ml) (FIG. 21B). *p<0.05,** p<0.01, *** p<0.001. Abbreviations: INF, inflammation (IFNγ-I1-1β);mM, millimolar; IGFBP3, insulin-like growth factor binding protein 3.

FIGS. 22A-22C. TMEM219 is expressed on human islets (FIGS. 22A-22C).*p<0.05, ** p<0.01, *** p<0.001. Abbreviations: β-ACT, beta actin.

FIGS. 23A-23D. TMEM219 is expressed on murine islets (FIG. 23A) and on amurine beta cell3 line (FIGS. 23B-23D). *p<0.05, ** p<0.01, *** p<0.001.Abbreviations: β-TC, murine beta cell line; β-ACT, beta actin.

FIGS. 24A-24E. IGFBP3 (50 ng/ml) increases apoptosis and caspase8expression (FIGS. 24A-24B) and reduces insulin release and expression(FIG. 24C; FIG. 24D, panels D1-D2; FIG. 24E) to a greater extent ascompared to pro-inflammatory stimuli (IFNγ-I1-1β) in a murine beta cellline in vitro. *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: IFNγ,interferon gamma; IL-1β, interleukin beta; IGFBP3, insulin-like growthfactor binding protein 3; β-TC, murine beta cell line.

FIGS. 25A-25C. IGFBP3 (50 ng/ml) increases apoptosis (FIG. 25A) andcaspase8 expression in murine islets (FIG. 25B with a reduction ofinsulin (FIG. 25C) in vitro. *p<0.05, ** p<0.01, *** p<0.001.

FIGS. 26A-26E. IGFBP3 (50 ng/ml) increases apoptosis and caspase8expression in human islets (FIGS. 26A-26B and FIG. 26C, panels C1-C2)and reduces insulin expression (FIGS. 26D, panels D1-D2, FIG. 26E) invitro. *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: Pi, PropidiumIodide; M30, monoclonal antibody M30 that recognizes caspase-cleavedcytokeratin 18; INS, insulin, IGFBP3, insulin-like growth factor bindingprotein 3.

FIG. 27A. IGFBP3 injection (150 μg/day for 15 days) in C57BL/6 micealters islet morphology in vivo after 8 weeks of diabetes (FIG. 27A,panels A1-A6). Abbreviations: STZ, streptozotocin; B6, naïve C57BL6/Jmice.

FIGS. 28A-28C. Ecto-TMEM219 (130 ng/ml) prevents IGFBP3-associatedapoptotic effects on murine beta cell line (FIGS. 28A-28B; FIG. 28C,panels C1-C3). *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: β-TC,murine beta cell line; INS, insulin, IGFBP3, insulin-like growth factorbinding protein 3.

FIGS. 29A-29B. Ecto-TMEM219 treatment (130 ng/ml) near-normalizescaspase 8 and insulin expression in murine islets in vitro. *p<0.05, **p<0.01, *** p<0.001. Abbreviations: IGFBP3, insulin-like growth factorbinding protein 3.

FIGS. 30A-30C. Ecto-TMEM219 (130 ng/ml) prevents IGFBP3-associatedapoptotic effects on human islets (FIGS. 30A-30B; FIG. 30C, panelsC1-C3). *p<0.05, ** p<0.01, *** p<0.001. Abbreviations: M30, monoclonalantibody M30 that recognizes caspase-cleaved cytokeratin 18; INS,insulin; IGFBP3, insulin-like growth factor binding protein 3.

FIGS. 31A-31C. Ecto-TMEM219 treatment (130 ng/ml) in diabetic micerescues serum insulin (FIG. 31A, FIG. 31C) and blood glucose levels (B).*p<0.05, ** p<0.01, *** p<0.001.

FIGS. 32A-32B. Working Hypothesis. Abbreviations: IGFBP3, insulin-likegrowth factor binding protein 3; IGF-I, insulin-like growth factor 1;IGF-IR, insulin-like growth factor 1 receptor.

DETAILED DESCRIPTION OF THE INVENTION Example 1

Material and Methods

60 individuals with long-standing T1D (T1D+ESRD) registered on thewaiting list for simultaneous pancreas-kidney transplantation (SPK) wereenrolled in the study and compared with 20 healthy subjects matched forage and gender (CTRL). Assessment of gastrointestinal symptoms,intestinal motility and intestinal mucosa pathology defined DE. CoSCswere identified on colonic purified crypts based on the expression ofCoSC specific markers (flow-cytometry, RT-PCR, Western Blot,transcriptome profiling). CoSCs self-renewal properties were assessed byevaluating the % of in vitro developed mini-guts and by characterizingtheir expression of cell lineages markers in different conditions (FIGS.15A-15F). Broad serum proteomic was used to detect circulating factorsthat may regulate CoSCs and candidate factors were then tested in the invitro mini-gut assay (FIG. 16). Detailed methods and statisticalanalysis are described below. The Study was approved by theInstitutional Review Board of Istituto di Ricovero e Cura a CarattereScientifico Ospedale San Raffaele, Milano, Italy (Enteropathy-PancreasKidneyTransplantation/01 Secchi/Fiorina).

Patients and Study Design

60 individuals with T1D+ESRD registered on the waiting list forsimultaneous pancreas-kidney transplantation (SPK) matched for (age 41to 43 years old), gender, and duration of T1D (29.4±1.8 years) wereenrolled in the study. 20 healthy subjects matched for age and gender(CTRL), with normal renal function and normal glycometabolic parameters,were studied as well. T1D+ESRD subjects were all on intensive insulintreatment at the time of enrollment in the study, while the CTRL groupwas not being administered any medication. All T1D+ESRD subjects were onthe same treatment as antiplatelet therapy (ASA) and anti-hypertension(angiotensin-converting-enzyme inhibitors), while 40 out of 60 receivedstatins when enrolled in the study. Subjects with clear signs ofinflammatory bowel diseases as well as celiac disease were not enrolled.

T1D+ESRD individuals were followed up for 8 years (mean follow-up:8.6±1.1 years) after receiving either SPK (n=30) or K+T1D (n=30)transplantation according to the macroscopic surgical evaluation at thetime of transplantation. Individuals taking an oral anticoagulant agentwere not included. SPK individuals were all insulin-independent for theentire follow-up period, whereas K+T1D individuals were on intensivesubcutaneous insulin therapy. All subjects provided informed consentbefore study enrollment. Studies not included in the routine clinicalfollow-up were covered by an appropriate Institutional Review Boardapproval (Enteropatia-trapianto/01 Secchi/Fiorina).

Transplantation and Immunosuppression

Organs for transplantation were obtained from deceased donors throughthe “North Italia Transplant” organ procurement consortium (NITp,Milan). After induction with ATG (thymoglobulin, IMTIX, SANGSTAT),immunosuppression was maintained using cyclosporine (through levelsbetween 100-250 ng/ml) or FK506 (through levels between 10-15 ng/ml),mycophenolate mofetil (500-2000 mg/day), and methylprednisolone (10mg/day). Steroids were withdrawn within 3-6 months aftertransplantation. All patients included in the T1D+ESRD and SPK groupswere on anti-platelet therapy (80% ASA and 20% ticlopidine) to preventgraft or fistula thrombosis. Metabolic status, renal function and bloodpressure were examined during enrolment and after transplantation every2 years thereafter. The estimate glomerular filtration rate (eGFR) wascalculated using the Modification of Diet in Renal Disease (MDRD)formula (Levey et al., 1999).

The Gastrointestinal Symptom Rating Scale (GSRS)

Gastrointestinal symptoms were evaluated by GSRS questionnaire inhealthy subjects, in long-standing T1D individuals (T1D+ESRD) and in SPKand K+T1D groups at 2, 4 and 8 years after transplantation. TheGastrointestinal Symptom Rating Scale (GSRS) is a questionnaireconsisting of 15 items with a seven-graded Likert scale defined bydescriptive anchors (Svedlund et al., 1988). The questionnaire wasoriginally constructed as an interview-based rating scale designed toevaluate a wide range of gastrointestinal symptoms and was latermodified to become a self-administered questionnaire. The higher thescores, the more severe the symptoms: the scale ranges from a minimumvalue of 1 to a maximum value of 7. If an individual's participation inthe study is discontinued, the value at the last available observationwill be carried forward in the analysis. The items can be grouped intofive dimensions previously identified on the basis of a factor analysis:abdominal pain syndrome (three items), reflux syndrome (two items),indigestion syndrome (four items), diarrhea syndrome (three items) andconstipation syndrome (three items).

Anorectal Manometry

Data on anorectal manometry were already available in healthy subjects,and were compared with those obtained by performing anorectal manometryin long-standing T1D individuals (T1D+ESRD) using a custom-designed,open-tip, 14-Fr diameter, PVC probe with seven lumens and a 4-cm latexballoon tied at the end of the probe (Bioengineering Laboratories Plc.,Milan, Italy) (Carrington et al., 2014; Remes-Troche et al., 2010). Thesphincter length was measured after a 10-minute run-in period, analpressure was recorded for 15 minutes in resting conditions. Subjectswere then instructed to squeeze the anus as tightly as possible and foras long as possible—for at least 20 seconds. Inventors' study evaluatedthe following items: Resting Tone, Contraction Tone, Reflex Response,and Urgency Response.

Pathology, Immunohistochemistry and Electron Microscopy

Colorectal endoscopy procedure was performed in healthy subjects, inlong-standing T1D individuals (T1D+ESRD) at baseline and in SPK andK+T1D groups at 2, 4, and 8 years after transplantation using a WelchAllyn optic sigmoid scope. Intestinal mucosal samples were fixed inbuffered formalin (formaldehyde 4% w/v and acetate buffer 0.05 M) androutinely processed in paraffin wax. 3 μm-thick sections of eachenrolled case were stained with Hematoxylin & Eosin (H&E) formorphological evaluations. For immunohistochemistry, 3 μm-thick sectionswere mounted on poly-L-lysine coated slides, deparaffinized and hydratedthrough graded alcohols to water. After antigen retrieval, performed bydipping sections in 0.01 M citrate buffer, pH 6 for 10 minutes in amicrowave oven at 650 W as well as endogenous peroxidase activityinhibition, performed by dipping sections in 3% hydrogen peroxide for 10minutes, incubation with primary antibodies was performed at 4° C. for18-20 hours, followed by the avidin-biotin complex procedure (Hsu etal., 1981). Immunoreactions were developed using 0.03%3,3′diaminobenzidine tetrahydrochloride, and then sections werecounterstained with Harris' hematoxylin. The following antibodies wereused: Ki67 (monoclonal, clone MIB1, 1:100 dilution, Dako, Carpinteria,Calif., USA), aldehyde dehydrogenase (monoclonal, clone 44/ALDH, 1:1000dilution, Transduction Laboratories, Franklin Lakes, N.J., USA), EphB2(monoclonal, clone 48CT12.6.4, 1:200 dilution, Lifespan Biosciences,Seattle, Wash., USA), LGR5 (monoclonal, clone 2A2, 1:100 dilution,Origene Technologies, Rockville, Md., USA), hTERT (monoclonal, cloneY182, 1:500 dilution, Millipore, Billerica, Mass., USA), glicentin(polyclonal, 1:1250 dilution, Milab, Malmo, Sweden), pancreaticpolypeptide (polyclonal, 1:500 dilution, Peninsula, Belmont, Calif.,USA), PYY (polyclonal, 1:1000 dilution, Biogenesis, Bournemouth, UK),serotonin (monoclonal, clone YCS, 1:50 dilution, Biogenesis),somatostatin (polyclonal, 1:550 dilution, Dako), IGF-I (polyclonal,1:500, Abcam) and IGF-1R (polyclonal, 1:100, Cell SignalingTechnologies), (Fiorina et al., 2003). For ultrastructural studies,samples were fixed for 2 hours at 4° C. in a mixture of 2%paraformaldehyde and 2% glutaraldehyde in 0.05 M cacodylate buffer, pH7.3. They were post-fixed in 1% osmium tetroxide for 1 hour at roomtemperature, then dehydrated and embedded in Epon-Araldite. Ultrathinsections were cut with a diamond knife and mounted on 200-mesh nickelgrids, previously coated with a Formvar film. Ultrathin sections werestained with aqueous uranyl acetate and Reynold's lead citrate solutionsand subsequently examined with a Philips Morgagni 268D electronmicroscope. Cases were grouped according to the number of neuroendocrinevesicles (n>3 and n<3) for statistical analysis. For crypt isolation,tissue was collected in a sample containing a mixture of antibiotics andprocessed as described in the next paragraph. The immunostainingintensity for EphB2 was graded as 1 (negative EphB2 gradient to fewcells positive per crypt per field) to 5 (strong EphB2 gradient in alllongitudinal crypts). An anti-IGFBP3 primary antibody (polyclonal, 1:50dilution, Sigma Aldrich) was immunohistochemically tested in liverbiopsies from patients with type 1 diabetes. Liver biopsies withoutpathological findings were used as controls. All of these tissue samplescame from the files stored at the Unit of Pathology of the Department ofBiomedical, Biotechnological, and Translational Sciences, University ofParma, Parma, Italy. The immunostaining intensity was graded as 1(mild), 2 (moderate), and 3 (strong), while its diffusion as 1 (focal),2 (zonal), and 3 (diffuse).

Immunoflurescence

Immunofluorescence samples obtained from liver biopsies were observedusing a confocal system (LSM 510 Meta scan head integrated with theAxiovert 200 M inverted microscope; Carl Zeiss, Jena, Germany) with a63× oil objective. Images were acquired in multitrack mode, usingconsecutive and independent optical pathways. The following primaryantibodies were used: rabbit IGFBP3 (1:10, Sigma) mouse Hep Par-1 (1:20,monoclonal, Dako), mouse CD163 (1:10, cloneMRQ26, CellMarque).

Mini-guts co-cultured with/without IGFBP3, with/without long-standingT1D serum+high glucose (35 mM Glucose) and those obtained from crypts ofT1D+ESRD individuals, were stained with Vimentin, Citocheratin 20,Aldheide Dehydrogenase and Synaptofisin for immunofluorescence analysisto assess expression of cell lineages markers (FIG. 15A, panels A1-A4;FIG. 15B, panels B1-B4; FIG. 15C, panels C1-C4; FIG. 15D, panels D1-D4;and FIG. 15D, panels E1-E4). The following primary antibodies were used:mouse vimentin (1:80, monoclonal, clone: V9 Dako) mouse Aldehyde(1:1000, monoclonal, clone: 44, BD), mouse citocherain 20 (1:100,monoclonal, clone:Ks20.8, Dako) and Synaptofisin (1:100, monoclonal,clone: syn88, BioGenex).

In Situ Hybridization

Paraffin sections of human colon mucosa were de-paraffinized andre-hydrated according to standard procedures. After treatment ofsections using 0.2M HCl for 15 minutes at room temperature, sectionswere washed 3 times in PBS and incubated for 15 min at 37° C. inproteinase K (30 μg/ml in PBS). 0.2% glycine in PBS was added for 1minute in order to neutralize Proteinase K activity, and samples werewashed twice in PBS. After post-fixation in 4% PFA for 10 min at roomtemperature and 3 washes in PBS, histone acetylation was achieved byincubating samples two times for 5 min in an aqueous solution containing1.5% triethanolamine, 0.15% HCl, and 0.6% acetic anhydride. Samples werethen washed and pre-hybridized for 1 hour at 68° C. in hybridizationsolution (50% formamide, 5×SSC, pH4.5, 2% Blocking Reagent (Roche),0.05% CHAPS (Sigma), 5 mM EDTA, 50 μg/ml Heparin (Sigma) and 50 μg/mlyeast RNA. For TMEM219, the digoxigenin-labelled probe was diluted 750ng/ml in hybridization solution and incubated for 24 hrs at 65° C.Post-hybridization washes were performed 3×20 min in 50% Formamide/2×SSCat 65° C. Sections were rinsed in TBS-T buffer (0.1M TrisHCl pH7.5,0.15M NaCl, 0.1% Tween20) and blocked for 30 min at room temperature inBlocking Solution (0.5% Blocking Reagent, 10% sheep serum in TBS-T).Sheep anti-DIG antibody (Fab fragment, Roche) was diluted 1/2000 inBlocking Solution and incubated overnight at 4° C. After this, sampleswere washed in TBS-T and then in NTM buffer (0.1M Tris pH9.5, 0.1M NaCl,0.05M MgCl2) and developed in NBT/BCIP solution (Roche) for 24 hrs.

CoSC Characterization

Crypt Purification

Muscle layer and sub-mucosa were carefully removed from human freshrectal biopsy specimens, and mucosa was incubated with a mixture ofantibiotics (Normocin, [Invivogen, San Diego, Calif. 92121, USA],Gentamycin [Invitrogen, Carlsbad, Calif., USA] and Fungizone[Invitrogen]) for 15 minutes at room temperature (RT). Next, tissue wascut into small pieces and incubated with 10 mM Dithiotreitol (DTT)(Sigma, St. Louis, Mo. 63103, USA) in PBS 2-3 times for 5 minutes at RT.Samples were then transferred to 8 mM EDTA in PBS and slowly rotated for60-75 minutes at 4° C. Supernatant was replaced by fresh PBS, andvigorous shaking of the sample yielded supernatants enriched in coloniccrypts. Fetal bovine serum (FBS, Sigma) was added to a finalconcentration of 5%, and fractions were centrifuged at 40×g for 2minutes in order to remove single cells. This washing procedure wasrepeated 3 times with Advanced DMEM/F12 (ADF, Gibco) medium supplementedwith 2 mM GlutaMax (Invitrogen), 10 mM HEPES (Sigma), and 5% FBS(Sigma).

200-300 isolated human colonic crypt units were mixed with 50 μlmatrigel and plated on pre-warmed 24-well culture dishes as alreadydescribed. After solidification (15-20 minutes at 37° C.), crypts wereoverlaid with 600 μl complete crypt culture medium [Wnt3a-conditionedmedium and Advanced DMEM/F12 (Life Technologies, Grand Island, N.Y.)50:50, supplemented with Glutamax, 10 mM HEPES, N-2 [1×], B-27 withoutretinoic acid [1×], 10 mM Nicotinamide, 1 mM N-Acetyl-L-cysteine, 50ng/ml human EGF (Life Technologies, Grand Island, N.Y.), 1 μg/ml RSPO1(Sino Biological, Beijing, China), 100 ng/ml human Noggin (Peprotech,Rocky Hill, N.J., USA), 1 μg/ml Gastrin (Sigma-Aldrich, St. Louis, Mo.),500 nM LY2157299 (Axon MedChem, Groningen, The Netherlands), 10 μMSB202190 (Sigma) and 0.01 μM PGE2 (Sigma)]. Medium was replaced everyother day. Rock inhibitor Y-27632 (10 μM, Sigma) was added to thecultures for the first 2-3 days. Purified crypts were directly culturedfor 8 days. Cell Lineages markers for enterocytes and enteroendocrinecells were assessed in the mini-guts and in the EphB2⁺ and EphB2⁻ sortedsingle cells with RT-PCR by testing: CHGA, KRT20 and EPCAM (LifeTechnologies, Grand Island, N.Y.). Colony forming efficiency (%) wasevaluated on freshly isolated crypts in order to exclude that thebioptic procedure and the isolation processing could have compromizedtheir efficiency in forming mini-guts in in vitro culture. DAPI stainingwas performed to confirm number of nuclei in freshly isolated cryptsfrom CTRL and T1D+ESRD subjects. Developed mini-guts with at least 1crypt domain were also counted and percentage was calculated in order toadd a more quantitative criteria to measure developed mini-guts (FIGS.17A-17G; 17M-17P). Insulin and glucose levels measured on long-standingT1D (T1D+ESRD) and CTRL serum are reported below:

Glucose levels (T1D+ESRD vs. CTRL, 178±47.5 vs 90±5.5 mg/dl, p0.0001);

Insulin levels (T1D+ESRD vs. CTRL, 12.9±4.6 vs 5.8±1.6 μIU/ml, p=0.009).

Flow Cytometry

The expression of the CoSC markers EphB2 (APC anti-human EphB2 antibody,R&D, Minneapolis, Minn.) and LGR5 (PE anti-human LGR5, Origene,Rockville, Md.) was determined by flow cytometry by excluding CD45- andCD11b-positive cells (V450 anti-human CD45 and CD11b, BD Biosciences,San Jose, Calif.). Propidium iodide (PI) was added (10 μg/ml) to excludedead cells. EphB2⁺ cells were also sorted by flow cytometry to obtain asingle cell suspension for culturing purposes. Intracellular detectionof human-tert (hTERT) was performed by permeabilizing cells and stainingwith primary anti-human hTERT antibody (GeneTex, Irvine, Calif.)followed by DAPI anti-goat secondary antibody (Life Technologies). Withregard to the analysis, cells were all first gated as PI⁻ before theassessment of other surface or intracellular markers. Samples were runon a BD LSR-Fortessa and analyzed by FSC Express 3.0 (DeNovo Software,Los Angeles, Calif., USA).

In Vitro Mini-Gut Generation Study

Crypts were isolated from healthy subject rectal biopsy samples andcultured as previously described to generate mini-guts. To createhyperglycemic conditions, the culturing medium was modified by addingglucose at different concentrations (35 mM: high glucose; 5 mM: normalglucose). To mimic uremic conditions, human uremic serum obtained fromlong-standing T1D individuals with ESRD was added to crypts, which werecultured as reported in the crypt culturing methods section. After 8days, crypts were collected, and the morphology, mini-gut growth,expression of intestinal signature markers (EphB2, LGR5, h-TERT), IGF-IRand TMEM219 (Life Technologies), and Caspase 9 (Life Technologies) wereexamined using RT-PCR. A pan-caspase inhibitor (caspase inhibitorZ-VAD-FMK, 20 mM, Promega, Madison, Wis.), a Caspase 8 selectiveinhibitor (Z-IETD-FMK, BD Pharmingen), a Caspase 9 selective inhibitor(Z-LEHD-FMK, BD Pharmingen), a caspase3 inhibitor Z-DEVD-FMK (BDPharmingen) were used in vitro in mini-guts to confirm the antiapoptoticeffect of IGFBP3.

To culture isolated crypts with crypts culturing medium containinghealthy subjects human serum, namely CTRL serum, in place of regularFBS, L-Wnt3 cells were grown in 10% CTRL serum to generate conditionedmedium that was further added 50:50 to Advanced DMEM/F12 medium in orderto obtain the crypts culture medium as previously described (see Cryptpurification).

To assess the properties of sorted EphB2⁺ cells in generating mini-guts,2000 sorted cells were mixed with 50 μl matrigel and plated onpre-warmed 24-well culture dishes. After solidification of the matrigel(10-15 min at 37° C.), cells were overlaid with “single cell growthmedium” (=complete crypt culture medium+10 M Rock inhibitor Y-27623).Medium was replaced with fresh single cell growth medium every otherday. Rock inhibitor was included in the culture medium for seven to ninedays.

Immunoblotting

Total proteins of intestinal bioptic samples were extracted in Laemmlibuffer (Tris-HCl 62.5 mmol/1, pH 6.8, 20% glycerol, 2% SDS, 5%β-mercaptoethanol) and their concentration was measured (Lowry et al.,1951). 35 μg of total protein was electrophoresed on 7% SDS-PAGE gelsand blotted onto nitrocellulose (Schleicher & Schuell, Dassel, Germany).Blots were then stained with Ponceau S. Membranes were blocked for 1 hin TBS (Tris [10 mmol/l], NaCl [150 mmol/l]), 0.1% Tween-20, 5% non-fatdry milk, pH 7.4 at 25° C., incubated for 12 h with 200 mg/ml of apolyclonal anti-goat EphB2 antibody or polyclonal anti-goat LGR5antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) ormonoclonal IGF-IR (Santa Cruz Biotechnology) and polyclonal TMEM219(R&D, Minneapolis, Minn.) diluted 1:200 or with a monoclonal mouseanti-β-actin antibody (Santa Cruz Biotechnology) diluted 1:1000 inTBS-5% milk at 4° C., washed four times with TBS-0.1% Tween-20, thenincubated with a peroxidase-labeled rabbit anti-goat IgG secondaryantibody (or rabbit anti mouse for β-actin) diluted 1:1000 (Santa CruzBiotechnology) in TBS-5% milk, and finally washed with TBS-0.1%Tween-20. The resulting bands were visualized using enhancedchemiluminescence (SuperSignal; Pierce, Rockford, Ill., USA).

Live Imaging of Intestinal Crypt Growth

Live imaging of mini-guts, obtained by purification and culture ofintestinal crypts of CTRL, T1D+ESRD and SPK individuals, was performedon a Zeiss Axiovert S100 equipped with environmental control (fromOko-Lab, Italy) with a chamber in which a humidified premixed gasconsisting of 5% CO₂ and 95% air was infused, and the whole setup wasset at 37° C. Images were acquired at 20-minute intervals for 72 hours.Images were acquired and processed using Time Lapse (Oko-Lab, Italy)and, if necessary, image editing was performed using Adobe PhotoshopElements 7.0.

Morphology Imaging Analysis

The images of mini-guts were taken at day 0, 5 and 8 days by invertedmicroscopy Leica DH/RB and acquired with Axio Vision AC Release 4.3.Pictures reported in figures represent mini-guts at day 5, 10×magnification.

Transcriptome Profiling

Total RNA was isolated from purified intestinal crypt suspension usingthe RNeasy Mini Kit (Qiagen, Valencia, Calif.) with on-column DNase Idigestion. Next, 3 μg total RNA from each sample was reverse-transcribedusing the RT2 First Strand kit (C-03; SABiosciences, Frederick, Md.).The inventors used the Human Stem Cell RT2 Profiler PCR Arrays(PAHS-405Z), the human Stem Cell Signaling PCR Array (PAHS-047Z,) and acustom array with the following genes: AXIN2, OLFM4, BMI1, RNF43, CDCA7,SLC12A2, CDK6, SOX9, DKC1, ZNRF3, ETS2, EPHB2, FAM84A, LGR5, GPX2, ACTB(SABiosciences). The Profiler PCR Arrays measure quantitatively theexpression of a panel of genes using SYBR Green-based real-time PCR(Kosinski et al., 2007). To assess the transcriptome profiling ofapoptotic markers and oxidative stress markers the Human Apoptosis PCRArrays (PAHS-012Z, SABiosciences) and the Human Oxidative Stress PCRArrays (PAHS-065Z, SABiosciences) were used.

qRT-PCR Analysis

RNA from purified intestinal crypts was extracted using Trizol Reagent(Invitrogen), and qRT-PCR analysis was performed using TaqMan assays(Life Technologies, Grand Island, N.Y.) according to the manufacturer'sinstructions. The normalized expression values were determined using theΔΔCt method. Quantitative reverse transcriptase polymerase chainreaction (qRT-PCR) data were normalized for the expression of ACTB, andΔΔCt values were calculated. Statistical analysis compared geneexpression across all cell populations for each patient via one-wayANOVA followed by Bonferroni post-test for multiple comparisons betweenthe population of interest and all other populations. Statisticalanalysis was performed also by using the software available RT² profilerPCR Array Data Analysis (Qiagen). For two groups comparison Student ttest was employed. Analysis was performed in triplicates after isolationof fresh crypts and/or after 8 days of culture of miniguts. Table I-Breports the main characteristics of primers used.

TABLE I-B Primers Gene Refseq Band Size Reference Symbol UniGene #Accession # (bp) Position LGR5 Hs.658889 NM_003667 91 1665 EPHB2Hs.523329 NM_004442 68 2908 TERT Hs.492203 NM_198253 106 1072 ACTBHs.520640 NM_001101 174 730 IGF-IR Hs.643120 NM_000875.3 64 2248 TMEM219Hs.460574 NM_001083613.1 60 726 KRT20 Hs.84905 NM_019010.2 75 974 CHGAHs.150793 NM_001275.3 115 521 EpcaM Hs.542050 NM_002354.2 95 784 LRP1Hs.162757 NM_002332.2 64 656 TGFbR1 Hs.494622 NM_001130916.1 73 646TGFbR2 Hs.604277 NM_001024847.2 70 1981 Caspase 8 Hs.599762NM_001080124.1 124 648 Caspase 9 Hs.329502 NM_001229.4 143 1405ELISA Assay

IGF-I and IGFBP3 levels in the pooled sera/plasma of all groups ofsubjects and in all groups of treated and untreated mice was assessedusing commercially available ELISA kits, according to the manufacturer'sinstructions (R&D and Sigma).

Human immortalized hepatoma cell line HuH-7 was cultured for 5 days inDMEM 10% FBS at different glucose concentrations: 5.5 mM, 20 mM and 35.5mM. Culturing supernatant was collected, and IGFBP3 was assessed usingan IGFBP3 ELISA kit (Sigma) according to the manufacturer'sinstructions. Collected cells were separated by trypsin and counted witha hemacytometer.

Insulin levels were assayed with a microparticle enzyme immunoassay(Mercodia Iso-Insulin ELISA) with intra- and inter-assay coefficients ofvariation (CVs) of 3.0% and 5.0%.

Recombinant Proteins and Interventional Studies

Recombinant human IGF-I (Sigma, 13769), (IGF-I), recombinant humanIGFBP3 (Life Technologies, 10430H07H5), (IGFBP3), and anti-IGF-IR(Selleckchem, Boston, OSI-906) were added to crypt cultures at day+2from isolation. IGFBP3 (Reprokine, Valley Cottage, N.Y.) wasadministered to naive and to STZ-treated B6 mice at 0.3 mg/mouse/day for15 days; IGF-I (Reprokine) and ecto TMEM219 were administered in vivo toSTZ-treated B6 mice after 2 weeks of diabetes at a dose of 5μg/mouse/day for 20 days and 100 μg/mouse/day for 15 days respectively.

Other molecules tested in in vitro mini-guts assay and added to cryptcultures at day +2 from isolation: Adiponectin (R&D), Thymosin β4(Abcam), C-reactive protein (Merck Millipore), Cystatin C (CellSignaling Technologies), Chromogranin A (Life Technologies),Fructose-bisphosphate aldolase (Novoprotein), Osteopontin (R&D),Ribonuclease pancreatic (RNASE, Novoprotein), Serum amyloid A protein(Abcam), Mannan-binding lectin serine protease 1 (MASP1, Novoprotein),Tumor necrosis factor-alpha (TNF-alpha, R&D), FaS Ligand (FasL, R&D).Hydrogen peroxide (H2O2, 50 μM) was also tested in the mini-guts assay.

Generation of Recombinant Human Ecto TMEM219

Recombinant human ecto-TMEM219 was generated using E. Coli as expressionhost for synthesis. Briefly, gene sequence of extracellular TMEM219 wasobtained:

(SEQ ID No. 2) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKL LTSEELALCGSR.

The DNA sequence of extracellular TMEM219 was cloned into a highcopy-number plasmid containing the lac promoter, which is thentransformed into the bacterium E. coli. Addition of IPTG (a lactoseanalog) activated the lac promoter and caused the bacteria to expressextracellular TMEM219 (ecto TMEM219). SDS-PAGE and Western Blot wereused to confirm purity higher than 90%. The molecular weight of the newgenerated protein recombinant human ecto TMEM219 was 80 kda.

Crypts from healthy subjects were isolated and cultured as previouslydescribed and ecto-TMEM219 was added to the culture at threeconcentrations (260 ng/ml, 130 ng/ml and 75 ng/ml) as compared to IGFBP3concentration used (2:1, 1:1 and 1:2) and appropriate controls were setup for each concentration. After 8 days of culture, caspase 8 and 9expression, CoSCs signature markers (EphB2 and LGR5) expression, numberof developed mini-guts, were further assessed.

Small RNA Interference

Isolated crypts obtained from healthy subjects were grown to generate invitro mini-guts in complete medium and in culturing medium modified byadding high glucose and long-standing T1D serum as previously described(see in vitro mini-gut generation study in online methods). After 72 hof culture, which allowed the crypts to recover, 750 ng of smallinterfering RNA (siRNA; Flexitube siRNA SI04381013, Qiagen, Valencia,Calif.) in 100 μl culture medium without serum and with 6 μl HiPerFectTransfection Reagent (Qiagen) were incubated at room temperature toallow for the formation of transfection complexes. Crypts were incubatedwith these transfection complexes under their normal growth conditionsfor 6 h. Analysis of gene silencing was performed at 24, 48 and 72 h byevaluating the percentage of normal mini-gut development. Control siRNAwas used as a negative control to confirm the effect of gene silencing.

Proteomic Analysis

8 μl of pooled serum from 10 patients per group were depleted using aProteoPrep 20 spin column (Sigma), thus allowing for the removal of the20 highly abundant proteins. The procedure was twice repeated in orderto obtain ˜99% depletion, according to the manufacturer's instructions.The recovered supernatant was analyzed to determine total proteinconcentration using the Direct Detect IR spectrophotometer and BSA as astandard. In order to obtain enough protein for proteomic analysis, 32μl from each pool were processed as above described. 40 μg of totalprotein from each sample was in-solution digested using the Filter AidedSample Preparation (FASP) protocol as reported in the literature(Wisniewski et al., 2009). Samples were desalted using C18 homemade tipcolumns (C18 Empore membrane, 3M) and injected into a capillarychromatographic system (EasyLC, Proxeon Biosystems, Thermo Scientific).Peptide separations were performed on a homemade 25 cm reverse phasespraying fused silica capillary column, packed with 3 μm ReproSil Pur120 C18-AQ. A gradient of eluents A (pure water with 2% v/v ACN, 0.5%v/v acetic acid) and B (ACN with 20% v/v pure water with 0.5% v/v aceticacid) was used to achieve separation (0.15 μL/minute flow rate) (from 10to 35% B in 230 minutes, from 35 to 50% B in 5 minutes and from 50 to70% B in 30 minutes). Mass spectrometry analysis was performed using anLTQ-Orbitrap mass spectrometer (Thermo Scientific, Waltham, Mass.)equipped with a nanoelectrospray ion source (Proxeon Biosystems). Fullscan mass spectra were acquired with the lock-mass option and resolutionset to 60,000. The acquisition mass range for each sample was from m/z300 to 1750 Da. The ten most intense doubly and triply charged ions wereselected and fragmented in the ion trap using a normalized collisionenergy 37%. Target ions already selected for the MS/MS were dynamicallyexcluded for 120 seconds. All MS/MS samples were analyzed using Mascot(v.2.2.07, Matrix Science, London, UK) search engine to search theUniProt_Human Complete Proteome_ cp_hum_2013_12. Searches were performedwith trypsin specificity, two missed cleavages allowed, cysteinecarbamidomethylation as fixed modification, acetylation at proteinN-terminus, and oxidation of methionine as variable modification. Masstolerance was set to 5 ppm and 0.6 Da for precursor and fragment ions,respectively. To quantify proteins, the raw data were loaded into theMaxQuant software version 1.3.0.5 (Cox et al., 2011). Label-free proteinquantification was based on the intensities of precursors. Peptides andproteins were accepted with an FDR less than 1%, two minimum peptidesper protein. The experiments were performed in technical triplicates.The complete dataset of proteins, obtained by proteomic analysis (TableI-C), was analyzed by Student's t-test using MeV software v. 4_8_1. 47proteins, which were significantly different (p-value <0.01) in controlpool versus T1D-ESDR pool, were further submitted to hierarchicalclustering analysis.

TABLE I-C List of quantified proteins identified by proteomic. analysisThe table reports correspondence between numbers and names of proteinsdetected by proteomic analysis Original row Protein names 1 14-3-3protein zeta/delta 4 Actin, cytoplasmic 1; Actin, cytoplasmic 1,N-terminally processed; Actin, cytoplasmic 2; Actin, cytoplasmic 2, N-terminally processed 5 Adiponectin 6 Afamin 8 Alpha-1-antichymotrypsin;Alpha-1-antichymotrypsin His-Pro- less 9 Alpha-1-antitrypsin; Shortpeptide from AAT 12 Alpha-2-HS-glycoprotein; Alpha-2-HS-glycoproteinchain A; Alpha-2-HS-glycoprotein chain B 13 Alpha-2-macroglobulin 14Alpha-actinin-1 16 Angiotensinogen; Angiotensin-1; Angiotensin-2;Angiotensin-3 17 Antithrombin-III 18 Apolipoprotein A-I; Truncatedapolipoprotein A-I 20 Apolipoprotein A-IV 21 Apolipoprotein B-100;Apolipoprotein B-48 22 Apolipoprotein C-I; Truncated apolipoprotein C-I23 Apolipoprotein C-II 24 Apolipoprotein C-III 25 Apolipoprotein C-IV 26Apolipoprotein D 28 Apolipoprotein F 29 Apolipoprotein L1 31Apolipoprotein(a) 34 Attractin 35 Basement membrane-specific heparansulfate proteoglycan core protein; Endorepellin; LG3 peptide 36Beta-2-glycoprotein 1 37 Beta-2-microglobulin; Beta-2-microglobulin formpI 5.3 39 Beta-Ala-His dipeptidase 42 C4b-binding protein beta chain 43Cadherin-1; E-Cad/CTF1; E-Cad/CTF2; E-Cad/CTF3 44 Cadherin-13 45Cadherin-5 46 Calreticulin 50 Carboxypeptidase N subunit 2 51 Cartilageoligomeric matrix protein 54 CD44 antigen 57 Ceruloplasmin 59Chromogranin-A; Vasostatin-1; Vasostatin-2; EA-92; ES-43; Pancreastatin;SS-18; WA-8; WE-14; LF-19; AL-11; GV-19; GR-44; ER-37 60 Clusterin;Clusterin beta chain; Clusterin alpha chain; Clusterin 62 Coagulationfactor V; Coagulation factor V heavy chain; Coagulation factor V lightchain 63 Coagulation factor X; Factor X light chain; Factor X heavychain; Activated factor Xa heavy chain 65 Cofilin-1 66 Collagenalpha-3(VI) chain 68 Complement C1r subcomponent; Complement C1rsubcomponent heavy chain; Complement C1r subcomponent light chain 71Complement C2; Complement C2b fragment; Complement C2a fragment″ 72Complement C3; Complement C3 beta chain; Complement C3 alpha chain; C3aanaphylatoxin; Complement C3b alpha chain; Complement C3c alpha chainfragment 1; Complement C3dg fragment; Complement C3g fragment;Complement C3d fragment; Complement C3f fragment; Complement C3c alphachain fragment 2 73 Complement C4-A; Complement C4 beta chain;Complement C4-A alpha chain; C4a anaphylatoxin; C4b-A; C4d-A; ComplementC4 gamma chain 74 Complement C4-B; Complement C4 beta chain; ComplementC4-B alpha chain; C4a anaphylatoxin; C4b-B; C4d-B; Complement C4 gammachain 75 Complement C5; Complement C5 beta chain; Complement C5 alphachain; C5a anaphylatoxin; Complement C5 alpha chain 76 Complementcomponent C1q receptor 77 Complement component C6 78 Complementcomponent C7 84 Complement factor D 88 Complement factor I; Complementfactor I heavy chain; Complement factor I light chain 89Corticosteroid-binding globulin 90 C-reactive protein; C-reactiveprotein(1-205) 91 Cystatin-C 92 Cystatin-M 95 EGF-containingfibulin-like extracellular matrix protein 1 96 Endothelial protein Creceptor 97 Extracellular matrix protein 1 98 Extracellular superoxidedismutase [Cu—Zn] 99 Fetuin-B 100 Fibrinogen alpha chain; FibrinopeptideA; Fibrinogen alpha chain 101 Fibrinogen beta chain; Fibrinopeptide B;Fibrinogen beta chain 102 Fibrinogen gamma chain 103 Fibronectin;Anastellin; Ugl-Y1; Ugl-Y2; Ugl-Y3 104 Fibulin-1 105 Ficolin-3 106Fructose-bisphosphate aldolase A; Fructose-bisphosphate aldolase 107Galectin-3-binding protein 108 Gamma-glutamyl hydrolase 109 Gelsolin 111Glyceraldehyde-3-phosphate dehydrogenase 112 Haptoglobin; Haptoglobinalpha chain; Haptoglobin beta chain 117 Heparin cofactor 2 122 Hypoxiaup-regulated protein 1 123 Ig alpha-1 chain C region 125 Ig gamma-1chain C region 126 Ig gamma-2 chain C region 127 Ig gamma-3 chain Cregion 129 Ig heavy chain V-II region SESS; Ig heavy chain V-II regionOU 130 Ig heavy chain V-III region BRO; Ig heavy chain V-III region TEI;Ig heavy chain V-III region BUT; Ig heavy chain V-III region WEA 134 Igheavy chain V-III region VH26 135 Ig kappa chain C region 136 Ig kappachain V-I region EU; Ig kappa chain V-I region CAR 142 Ig kappa chainV-III region WOL; Ig kappa chain V-III region SIE; Ig kappa chain V-IIIregion Ti; Ig kappa chain V-III region GOL 144 Ig kappa chain V-IVregion Len 145 Ig lambda chain V-I region HA; Ig lambda chain V-I regionWAH; Ig lambda chain V-II region MGC; Ig lambda chain V-II region WIN146 Ig lambda chain V-III region LOI 148 Ig lambda-2 chain C regions; Iglambda-3 chain C regions; Ig lambda-6 chain C region 153 Immunoglobulinlambda-like polypeptide 5; Ig lambda-1 chain C regions 154 Insulin-likegrowth factor-binding protein 2 155 Insulin-like growth factor-bindingprotein 3 156 Insulin-like growth factor-binding protein 6 158Inter-alpha-trypsin inhibitor heavy chain H1 159 Inter-alpha-trypsininhibitor heavy chain H2 160 Inter-alpha-trypsin inhibitor heavy chainH3 161 Inter-alpha-trypsin inhibitor heavy chain H4; 70 kDa inter-alpha-trypsin inhibitor heavy chain H4; 35 kDa inter-alpha-trypsin inhibitorheavy chain H4 164 Keratin, type I cytoskeletal 10 165 Keratin, type Icytoskeletal 9 166 Keratin, type II cytoskeletal 1 167 Kininogen-1;Kininogen-1 heavy chain; T-kinin; Bradykinin; Lysyl-bradykinin;Kininogen-1 light chain; Low molecular weight growth-promoting factor168 Leucine-rich alpha-2-glycoprotein 171 L-lactate dehydrogenase Bchain; L-lactate dehydrogenase 174 Lumican 175 Lymphatic vesselendothelial hyaluronic acid receptor 1 176 Lysozyme C 178 Mannan-bindinglectin serine protease 1; Mannan-binding lectin serine protease 1 heavychain; Mannan-binding lectin serine protease 1 light chain 180 Monocytedifferentiation antigen CD14; Monocyte differentiation antigen CD14,urinary form; Monocyte differentiation antigen CD14, membrane-bound form181 Multimerin-1; Platelet glycoprotein Ia*; 155 kDa platelet multimerin183 Neudesin 185 Neural cell adhesion molecule L1-like protein;Processed neural cell adhesion molecule L1-like protein 187 Osteopontin188 Peptidase inhibitor 16 189 Peptidyl-prolyl cis-trans isomerase A;Peptidyl-prolyl cis-trans isomerase 192 Phosphatidylethanolamine-bindingprotein 4 194 Pigment epithelium-derived factor 197 Plasminogen; Plasminheavy chain A; Activation peptide; Angiostatin; Plasmin heavy chain A,short form; Plasmin light chain B 198 Platelet basic protein; Connectivetissue-activating peptide III; TC-2; Connective tissue-activatingpeptide III(1-81); Beta- thromboglobulin; Neutrophil-activating peptide2(74); Neutrophil-activating peptide 2(73); Neutrophil-activatingpeptide 2; TC-1; Neutrophil-activating peptide 2(1-66);Neutrophil-activating peptide 2(1-63) 199 Platelet glycoprotein Ib alphachain; Glycocalicin 200 Plexin domain-containing protein 2 203Profilin-1 204 Proline-rich acidic protein 1 205 Properdin 206Prostaglandin-H2 D-isomerase 207 Protein AMBP; Alpha-1-microglobulin;Inter-alpha-trypsin inhibitor light chain; Trypstatin 209 Prothrombin;Activation peptide fragment 1; Activation peptide fragment 2; Thrombinlight chain; Thrombin heavy chain 212 Receptor-type tyrosine-proteinphosphatase gamma 213 Retinol-binding protein 4; Plasma retinol-bindingprotein(1-182); Plasma retinol-binding protein(1-181); Plasmaretinol-binding protein(1-179); Plasma retinol-binding protein(1-176)214 Rho GDP-dissociation inhibitor 2 215 Ribonuclease pancreatic 216Scavenger receptor cysteine-rich type 1 protein M130; Soluble CD163″ 217Secreted and transmembrane protein 1 221 Serotransferrin 222 Serumalbumin 223 Serum amyloid A protein 225 Serum amyloid P-component; Serumamyloid P-component(1- 203) 226 Serum paraoxonase/arylesterase 1 228SPARC-like protein 1 230 Talin-1 232 Tenascin-X 233 Tetranectin 234Thrombospondin-1 235 Thrombospondin-4 236 Thymosin beta-4; Hematopoieticsystem regulatory peptide 237 Thyroxine-binding globulin 239Transgelin-2 240 Trans-Golgi network integral membrane protein 2 242Tropomyosin alpha-4 chain 243 Vascular cell adhesion protein 1 244Vasorin 245 Vinculin 247 Vitamin K-dependent protein C; VitaminK-dependent protein C light chain; Vitamin K-dependent protein C heavychain; Activation peptide 248 Vitamin K-dependent protein S 249 VitaminK-dependent protein Z 250 Vitronectin; Vitronectin V65 subunit;Vitronectin V10 subunit; Somatomedin-B 251 von Willebrand factor; vonWillebrand antigen 2 254 Zinc-alpha-2-glycoprotein 258 Vitamin D-bindingprotein 259 Complement factor H 266 Fibulin-1 267 Mannan-binding lectinserine protease 1 270 Complement factor H-related protein 4Strategy to Select Candidate Proteins

Among the 46 factors that segregated separately in long-standing T1Dsubjects and healthy controls, the inventors first selected those with amore significant difference in LFQ intensity in comparing the two groups(p>0.005), leading to the exclusion of 12 factors (FIG. 16). Next, theinventors evaluated whether altered factors may be associated withintestinal disorders and/or with the development of diabetes bysearching for already reported studies and publications in the field.This led us to exclude other 12 factors. The inventors also excludedthose factors mainly related to the lymphoid compartment (n=5). Theinventors ended up with 17 factors. The inventors excluded cell-membraneproteins (n=4) and proceeded with testing the remaining (n=13) in themini-gut assay. Two factors were not available to be tested in vitro.The inventors tested n=11 proteins in total.

Animal Studies

C57BL/6 (B6) mice were obtained from the Jackson Laboratory, Bar Harbor,Me. All mice were cared for and used in accordance with institutionalguidelines approved by the Harvard Medical School Institutional AnimalCare and Use Committee. Mice were rendered diabetic with streptozotocininjection (225 mg/kg, administered i.p.; Sigma). Diabetes was defined asblood glucose levels >250 mg/dL for 3 consecutive measures. Diabeticenteropathy was assessed as follows: briefly, the entire intestine wasextracted from sacrificed mice and flushed with PBS. The extreme part ofthe colon was then cut and divided in two pieces. One piece of colontissue was directly submerged in formalin while the other was cutlongitudinally to expose the lumen and the internal mucosa and thensubmerged in formalin. Tissue was then paraffin embedded and processedfor H&E and immunostaining. In addition, colonic tissue was also cut andisolation of colonic stem cells was performed as previously described(Merlos-Suarez et al., 2011). Briefly, colon was cut into 2-4 mm piecesand the fragments were washed in 30 mL ice-cold PBS. Fragments were thetransferred in 50 ml tubes containing pre-warmed 20 mM EDTA-PBS andincubated at 37° C. for 30 min. After incubation the suspended tissuewas transferred into tube containing 30 ml cold PBS and centrifuged.Crypts were resuspended in 13 ml cold DMEMF12, washed with PBS anddigested in 5-10 ml of trypsin/DNAse solution at 37° C. for 30 min.Crypts were then resuspended in DMEMF12/EDTA, filtered in 40 micronstrainer twice and washed. Finally, crypts were then resuspended in flowmedium (DMEM+FBS+EDTA) and stained for anti EphB2-APC (R&D), mouseanti-CD45-PeRCP and mouse anti-CD11b-PE (BD Pharmingen). Samples wererun using a FACSCalibur Analyzer and data analyzed with FlowJo.

Part of the tissue was also snap frozen and stored in Tryzol to performRT-PCR studies for the following markers:

Gene Refseq Band Size Reference Symbol: UniGene #: Accession #: (bp):Position: LGR5 Mm.42103 NM_010195.2 64 571 EPHB2 Mm.250981 NM_010142.285 1696 Casp8 Mm.336851 NM_001080126.1 96 1525 Casp9 Mm.88829NM_001277932.1 68 377 GAPDH Mm. 304088 NM_008084.2 107 75

Finally, plasma and serum were collected to perform analysis of IGF-I(IGF-I ELISA kit, R&D), IGFBP3 (IGFBP3 ELISA kit, R&D) and insulinlevels (Mercodia Mouse Insulin ELISA kit). Blood glucose was monitoredtwice a week for the 8 weeks in order to confirm diabetes onset andpermanence.

Statistical Analysis

Data are presented as mean and standard error of the mean (SEM) and weretested for normal distribution with the Kolmogorov-Smirnov test and forhomogeneity of variances with Levene's test. The statisticalsignificance of differences was tested with two-tailed t-test and thechi-square (χ2) tests. Significance between the two groups wasdetermined by two-tailed unpaired Student's t test. For multiplecomparisons, the ANOVA test with Bonferroni correction was employed. Alldata were entered into Statistical Package for the Social Science(SPSS®, IBM®, SPSS Inc., Chicago, Ill.) and analyzed. Graphs weregenerated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla,Calif.). All statistical tests were performed at the 5% significancelevel.

Results

Intestinal Dysfunction and Clinical Symptoms are Present inLong-Standing T1D

The inventors first characterized intestinal morphology and function ina population of individuals with long-standing T1D and end stage renaldisease (T1D+ESRD) and in healthy subjects (CTRL). Severe intestinalsymptoms, such as diarrhea, abdominal pain and constipation, wereevident in T1D+ESRD individuals as assessed using the GastrointestinalSymptom Rating Scale (GSRS) questionnaire (FIGS. 1A-1C). Symptoms wereassociated with abnormalities in anorectal sphincter function (FIGS.1D-1F). The intestinal mucosa was altered in individuals with T1D+ESRDas compared to healthy subjects, with lower number of crypts, distortionand zonal sclerosis of the lamina propria (FIG. 1G, panels G1-G2, FIG.1H). A significant reduction in epithelial cell proliferation asassessed by Ki67 (MIB1 antibody) staining (FIG. 1I, panels I1-I2, FIG.1J), signs of neural degeneration (FIG. 1K, panels K1-K2, FIG. 1L) andreduction in serotonin expression in intestinal neuroendocrine cells(FIGS. 1M, panels M1-M2, FIG. 1N) were observed, confirming the presenceof DE in these individuals.

CoSCs are Altered in Long-Standing T1D

The characterization of colonic crypts, revealed a significant reductionin EphB2⁺ expression and in the number of aldehyde dehydrogenase (Aldh)⁺immunoreactive cells, both markers of local stem cells (Carpentino etal., 2009; Jung et al., 2011), in T1D+ESRD individuals as compared tohealthy subjects (FIG. 1O, panels O1-O2; FIG. 1P; FIG. 1Q, panels Q1-Q2,R). A profound decrease was evident, upon gating on PI⁻ cells at FACSanalysis (FIGS. 8A-8C), in the percentage of EphB2^(hi),EphB2^(hi+)LGR5⁺ and EphB2⁺h-TERT⁺ cells isolated from intestinal cryptsobtained from T1D+ESRD individuals as compared to healthy subjects(FIGS. 2A-2B, FIGS. 2C-2E, FIGS. 8D-8E) and was confirmed by RT-PCR(FIG. 2: F-H) and western blot (WB) analysis (FIG. 8F). Transcriptomeprofiling of crypts obtained from T1D+ESRD documented a decreasedexpression of Notch pathway (Notch1 and 2, JAG1, Dll1, Sox1 and 2), Wntpathway (APC, FZD1, DKC1, ETS2, FAM84A, GPX2, RNF43) and BMP pathway(BMP1, BMP2, BMP3) genes, previously known pathways that control CoSCs,as compared to the expression of these genes in healthy subjects (FIG.8G and Table II).

TABLE II List of up and down regulated stem cell target genes identifiedby transcriptomic profiling in CTRL vs. T1D + ESRD freshly isolatedcolonic crypts (at least p < 0.05). Down-regulated genes Up-regulatedgenes ACTC1 APC CD44 DVL1 BTRC SOX1 SOX2 WNT1 CCND2 FZD1 ADAR ACAN ALPICD8A COL1A1 COL2A1 COL9A1 BMP1 BMP2 BMP3 CCNA2 CCNE1 CDC42 CDK1 CTNNA1CXCL12 PARD6A CD3D CD8B MME CD4 DLL1 HDAC2 NOTCH1 DLL3 JAG1 NOTCH2 DTX2KAT2A NUMB EP300 FGF2 FGF3 FGFR1 GDF3 ISL1 KRT15 MSX1 MYOD1 T GJA1 GJB1GJB2 KAT8 RB1 h-TERT NCAM1 SIGMAR1 TUBB3 ABCG2 ALDH1A1 PDX1 IGF-I DHHBGLAP

Analysis of—CoSC signature genes revealed that LGR5, EphB2 (Gracz etal., 2013; Merlos-Suarez et al., 2011), h-TERT (Breault et al., 2008)and other intestinal stem cell marker genes (Hughes et al., 2011; Munozet al., 2012; Ziskin et al., 2013) were significantly underexpressed inT1D+ESRD as compared to healthy subjects as well (FIG. 2I), confirmingthat the CoSCs are altered in individuals with DE.

In Vitro Generation of Mini-Guts is Altered in Long-Standing T1D

In order to evaluate CoSC self-renewal properties, the inventors usedthe in vitro mini-gut assay. Indeed, crypts isolated from T1D+ESRDindividuals and cultured in vitro for 8 days formed small spheroidmini-guts that failed to grow as compared to healthy subjects (FIG. 2J,panels J1-J2; FIG. 2K), despite a comparable viability (FIG. 8: H-I) andefficiency of forming mini-guts in both groups (FIG. 8J). To begin toelucidate the effect of circulating factors and high glucose on CoSCs,the inventors cultured isolated intestinal crypts obtained from healthysubjects in high glucose with/without serum obtained from long-standingT1D individuals in vitro for 8 days (FIG. 2L, panels L1-L4; FIG. 2M).High glucose partially prevented the generation of fully maturemini-guts and synergized with serum of long-standing T1D individuals inaltering CoSC self-renewal properties, such that mini-guts appearedcollapsed (FIG. 2L, panels L2-L4). Analysis of gene expression alsorevealed changes in the CoSC signature (FIG. 2N), thus suggesting thathyperglycemia and circulating factors act together to alter CoSCregenerative properties in long-standing T1D.

Serum Unbiased Proteomic Profiling Revealed Increased Levels of IGFBP3in Long-Standing T1D

In order to identify potential circulating factors that may serve asenterotrophic hormones and may have a role in regulating the CoSCs, theinventors compared the serum proteome of healthy subjects with T1D+ESRDindividuals using an unbiased proteomic array. A clear proteomic profilewas evident in T1D+ESRD individuals as compared to healthy subjects,with more than 50% of the detected proteins segregating in either onegroup or the other (FIG. 3A). Some proteins were associated withdiabetes, and some were growth factors or stem cell-related proteins orwere potentially involved in intestinal functions (FIG. 3A). Inparticular, the levels of IGF-I binding proteins (IGFBP2 and 3) weredetectable in long-standing T1D individuals as compared to healthysubjects, with IGFBP3 almost 5-fold increased (FIG. 3B), while IGFBP1,4, 5 and 6 remained almost undetectable. Interestingly, in the liver ofindividuals with long-standing T1D, hepatocytes, but not Kuppfer cells,showed a higher IGFBP3 immunohistochemical expression as compared tohealthy subjects (FIG. 3C, panels C1-C2; FIG. 8K, FIG. 8L; panelsL1-L6), suggesting an increase in IGFBP3 hepatic synthesis and release.The effect of high glucose on IGFBP3 hepatic release was confirmed bythe detection of increased IGFBP3 levels in the supernatant of humanimmortalized hepatocytes exposed to high glucose (FIG. 3D). Finally,serum levels of free IGF-I appeared significantly reduced inlong-standing T1D as compared to healthy subjects (FIG. 3E), indicatingthat circulating IGF-I and IGFBP3 levels are altered in long-standingT1D.

Peripheral IGFBP3 and IGF-I Control CoSCs

To further elucidate the role of circulating IGF-I and IGFBP3 in theregulation of the CoSCs and of intestinal epithelial proliferation, theinventors demonstrated the expression of IGF-IR and of IGFBP3 receptor(TMEM219) on isolated crypts (FIG. 3F; FIG. 3H; FIG. 8M; FIG. 8N, panelsN1-N2), using RT-PCR and WB (FIGS. 3F, 3H, 8M), and confirmed theexpression of IGF-IR on CoSCs with immunostaining (FIG. 8N, panelsN1-N2), and of TMEM219 with in situ hybridization (FIG. 3G, panelsG1-G2). In order to mechanistically confirm the role of IGF-I and IGFBP3on CoSC, the inventors tested the effect of several molecules,identified by proteomic profiling, in their in vitro mini-gut assay.Inventors' strategy to select potential targets is reported inSupplemental Information. The severely altered mini-guts generated fromintestinal samples obtained from T1D+ESRD individuals were rescued bythe addition of recombinant human IGF-I (IGF-I) to the culture medium(FIG. 3I), while the addition of recombinant human IGFBP3 (IGFBP3)resulted in the abrogation of the positive effect observed with IGF-I,with a decreased development of mini-guts and increased formation ofcollapsed and distorted organoids (FIG. 3I). Because IGFBP3 has beenrecently shown to act independently of IGF-I (Williams et al., 2007) viathe IGFBP3 receptor (TMEM219)(Baxter, 2013), it was necessary to clarifywhether IGFBP3 exerts its effects on CoSCs by binding IGF-I or bydirectly targeting TMEM219 on CoSCs. The inventors first confirmed thatIGFBP3 has a direct pro apoptotic effect on CoSCs by demonstratingincreased Caspase 8 and 9 expression in mini-guts obtained from healthysubjects and long-standing T1D individuals and cultured with IGFBP3(FIG. 3J, FIGS. 9A-9B), while the addition of a Pan-Caspase inhibitor(Z-VAD-FMK) or the addition of both selective inhibitors of caspases 8and 9, but not that of other caspase cascade inhibitors (Caspase 3inhibitor) abrogated the IGFBP3 effect (FIG. 3K). The inventors thendemonstrated that the addition of IGF-I did not rescue the developmentof mini-guts obtained from healthy subjects and exposed to IGFBP3 (FIG.3L), confirming that IGFBP3 may act through both a direct and indirectIGF-I mechanism. Interestingly, high glucose alone was unable tocompletely disrupt mini-gut growth, and anti-IGF-IR did not worsengrowth and morphology of mini-guts formed from healthy subjects (FIG.3L). The addition of IGF-I to mini-guts generated from healthy subjects,but cultured with high glucose and serum from long-standing T1Dindividuals, rescued mini-gut morphology, while IGFBP3 abolished thepositive effect of IGF-I when added to the mini-gut culture (FIG. 3L).Interestingly, the use of healthy subjects “CTRL” serum in culturingcrypts obtained from long-standing T1D nearly restored mini-gutsdevelopment/morphology, indicating that circulating factors, and inparticular IGF-I/IGFBP3 dyad, control CoSCs (FIG. 9C-9D). The inventorsthen genetically modulated TMEM219 expression by using siRNA in vitro inmini-guts obtained from healthy subjects. Knockdown of TMEM219 inmini-guts preserved their ability to grow and self-renew, despite theaddition of IGFBP3 and high glucose with long-standing T1D serum (FIG.3M). Concomitant blockade of TMEM219 by SiRNA and IGF-IR by blockingantibody did not result in any additional beneficial effect on mini-gutsdevelopment despite using serum from healthy subjects or fromlong-standing T1D (FIG. 9E).

Other circulating proteins, which appeared altered in serum proteome oflong-standing T1D individuals, were tested in the in vitro mini-gutassay and did not show any significant effect on mini-guts growth (FIGS.9F-9G). C-peptide and insulin, whose levels are commonly altered in T1Dand which may interfere with IGF-I/IGFBP3 dyad by binding IGF-IR (FIG.9H), were tested as well and did not show any effect.

To further confirm that IGF-I/IGFBP3 dyad targets effectively CoSCs andnot only crypts, the inventors tested its effect on single cell-derivedmini-guts. The inventors flow sorted EphB2⁺ cells from isolated cryptsand established that TMEM219 was highly expressed on their surface (FIG.4A). The inventors then cultured EphB2⁺ cells in the in vitro singlecell-derived mini-gut assay and confirmed that high glucose andlong-standing T1D serum exposure as well as addition of IGFBP3significantly abrogate single cell-derived mini-guts growth, thusrecapitulating the main features reported in their previous observationson crypt-derived mini-guts (FIG. 4B; FIG. 10A, panels A1-A3). Moreover,expression of Caspase 8 and 9 was up regulated in IGFBP3-treatedmini-guts and in those exposed to high glucose and long-standing T1Dserum, while Ki67, marker of proliferation, was significantly underexpressed (FIGS. 10B-10D).

Effect of the IGF-I/IGFBP3 Dyad on Previously Known Pathways thatControl CoSCs

In order to clarify the effects of IGF-I/IGFBP3 dyad on pathwayspreviously known to be involved in CoSC niche function (i.e.Wnt/Notch/BMP), the inventors obtained from their stem celltranscriptome profile the expression of niche specific gene transcripts.IGF-I restores significantly the expression of some factors associatedwith Wnt/Notch signaling pathways on mini-guts obtained from crypts ofT1D+ESRD (FIG. 10E, Table III), while IGFBP3 poorly affectsWnt/Notch/BMP gene expression in mini-guts obtained from crypts ofhealthy subjects or from those of T1D+ESRD (FIG. 10F, Table III).

TABLE III List of up and down-regulated stem cell target genesidentified by transcriptomic profiling in colonic crypts obtained fromCTRL and from T1D + ESRD and cultured with/without IGFBP3 and IGF-I (atleast p < 0.05). Down-regulated genes Up-regulated genes CTRL + IGF-ICD44, CDH1, COL9A1 ACAN, COL2A1, DLL1, FGF2, vs. FGF3, GDF3, GJA1,IGF-I, ISL1, CTRL MME, MSX1, NCAM1, NOTCH2, PDX1, SOX1, SOX2, h-TERTCTRL + IGFBP3 CD8B, COL9A1, RB1, SOX1, h-TERT ASCL2, COL2A1, DHH, DLL1,vs. DTX1, DVL1, FGF3, FGF4, CTRL FOXA2, FRAT1, GDF2, HSPA9, IGF1, KAT2A,MSX1, MYC, NEUROG2, S100B, WNT1 T1D + ESRD + IGF-I ACTC1, CD3D, CD4,COL9A1, DTX1, ABCG2, ADAR, BMP1, BMP2, vs. FGFR1 BTRC, CDC42, CTNNA1,T1D + ESRD CXCL12, DLL1, DTX2, GDF3, HDAC2, ISL1, JAG1, NOTCH1, NOTCH2,NUMB, PARD6A, PDX1, RB1, SIGMAR1, h-TERT T1D + ESRD + IGFBP3 ABCG2,ALDH1A1, ALPI, CD3D, CD4, ASCL2, KAT2A, MYC, NCAM1, vs. CD44, CD8A,CDC42, FGF2, FGFR1, NEUROG2, SOX2 T1D + ESRD JAG1, SIGMAR1, SOX1, TUBBAbbreviations: IGF-I, insulin-like growth factor 1; IGFBP3, insulin-likegrowth factor binding protein 3, CTRL, healthy subjects, T1D, type 1diabetes, ESRD, end-stage renal disease.

This confirms that IGF-I preserves the expression of some genes involvedin Wnt/Notch/BMP signaling, but also that IGFBP3 acts independently onCoSCs, without major alterations in the expression of key-target genesof the other previously known pathways.

Effect of IGF/IGFBP3 Dyad on Apoptotic Pathways in CoSCs

An extensive transcriptome analysis performed to clarify the IGFBP3caspase-mediated effect on mini-guts, (FIGS. 4C-4D, FIG. 10G-10H, TableIV), showed that addition of IGFBP3 to mini-guts grown from healthysubjects crypts, was associated with a significant up regulation ofcaspase-cascade activators (Caspases 8 and 9) and proapoptotic genes,while the anti-apoptotic gene Bcl2 was down regulated (FIG. 4C).

TABLE IV List of up and down-regulated pro/anti-apoptotic target genesidentified by transcriptomic profiling in CTRL vs. T1D + ESRD freshlyisolated colonic crypts and in those cultured with IGFBP3 and IGF-I (atleast p < 0.05). Down-regulated genes Up-regulated genes T1D + ESRDBCL2, NOL3, FAS CASP1, CASP10, CASP14, CASP5, vs. CASP6, CASP7, CASP8,CASP9, CTRL CD27, CRADD, FADD, FASLG, HRK, TNFRSF10A, TNFRSF10B,TNFRSF11B, TNFRSF1A, TNFRSF1B, TNFRSF25, TNFRSF9, TNFSF8, TRADD, TRAF3CTRL + IGF-I BNIPL3 CASP14, CASP5, CD27, CRADD, vs. FASLG, TNFRSF25,TNFSF8, CTRL TRADD CTRL + IGFBP3 BAX, BCL2 CASP5, CASP8, CASP9, FAS, vs.TNFRSF1B, TNFSF8, TRADD, CTRL TRAF3 T1D + ESRD + IGF-I CASP1, CASP10,CASP5, BCL2 vs. CASP6, CASP7, CASP8, T1D + ESRD CASP9, CRADD, FADD,TNFRSF11B, TNFRSF9, TNFSF8, TRADD, TRAF3 T1D + ESRD + IGFBP3 BAX, BCL2,NOL3, CASP9, CD27 vs. TNFRSF1B T1D + ESRD Abbreviations: IGF-I,insulin-like growth factor 1; IGFBP3, insulin-like growth factor bindingprotein 3, CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stagerenal disease.

Interestingly, anti-apoptotic genes (Bcl2, Fas, Nol3) were significantlyunderexpressed in mini-guts grown from T1D+ESRD crypts as well, ascompared to healthy subjects, while the majority of caspases relatedgenes (Caspase 1, 5, 7, 8, 9, 14) were over expressed (FIG. 10G).Moreover, the expression of genes involved in other pro apoptoticpathways was either not altered (i.e. Fas Ligand, FADD, TNF) orinhibited (TRADD) in T1D+ESRD mini-guts. The opposite effect wasobserved by adding IGF-I (FIG. 4D, FIG. 10H). The absence of alterationsin the expression of oxidative stress target genes (Table V) and of anyeffect of oxidative stress factors (FIGS. 10I-10J), confirmed the mainapoptotic-related caspase-mediated IGFBP3 mechanism whereby circulatingIGFBP3 directly controls CoSCs (FIG. 4E).

TABLE V List of up and down-regulated oxidative stress target genesidentified by transcriptomic profiling in CTRL vs. T1D + ESRD freshlyisolated colonic crypts and in those cultured with IGFBP3 and IGF-I (atleast p < 0.05). Down-regulated genes Up-regulated genes T1D + ESRDDUOX1, PRDX4, STK25, GSS CYBB, GPX5, KRT1, MT3, NOX4, vs. OXR1, PTGS1,SFTPD CTRL CTRL + IGF-I DUOX1, TXNRD AOX1, FTH1, GPX7, GSS, KRT1, vs.LPO, MPO, NCF1, NOS2, NOX4, CTRL OXR1, PTGS1, PTGS2, SCARA3, SFTPD, TPO,TTN CTRL + IGFBP3 NCF1, SOD3 AOX1, GPX5, GPX7, HSPA1A vs. KRT1, MB, MPO,NOX5, OXR1, CTRL PTGS1, SFTPD, TPO, TTN, TXNRD2, UCP2 T1D + ESRD + IGF-IDUOX1, EPHX2, MB, MT3, MPO, PRDX4, PRNP, STK25 vs. NCF1, OXR1, PTGS1,T1D + ESRD SOD3, SRXN1 T1D + ESRD + IGFBP3 CYBB, DUOX1, EPHX2 NOS2,STK25 vs. GPX3, GSTP1, HSPA1A T1D + ESRD MGST3, NCF1, NQO1, PRDX6, RNF7,TXN Abbreviations: IGF-I, insulin-like growth factor 1; IGFBP3,insulin-like growth factor binding protein 3, CTRL, healthy subjects,T1D, type 1 diabetes, ESRD, end-stage renal disease.Manipulation of the Circulating IGF-I/IGFBP3 Dyad Alters the Course ofDiabetic Enteropathy in a Preclinical Model

In order to further demonstrate the relevance of IGF-I/IGFBP3circulating factors in vivo, the inventors tested the effects of IGF-Iand IGFBP3 administration in a preclinical model of DE. After 8 weeks ofchemically-induced diabetes (using streptozotocin [STZ]), C57BL/6 (B6)mice showed a reduced number of crypts in the colorectal tissue (FIG.4F), which displayed increased depth and width in more than 70% of cases(FIG. 4G; FIG. 4H, panels H1-H3; FIG. 4I) and a reduced number of Aldh⁺cells (FIG. 4J; FIG. 4K, panels K1-K2). Interestingly, those mice showedincreased serum levels of IGFBP3 and low levels of IGF-I, with lowermurine insulin levels as compared to naïve B6 (FIGS. 11A-11C).Intraperitoneal (i.p.) administration of IGFBP3 in naïve B6 miceresulted in a reduction in local crypt numbers (FIG. 4F, FIG. 4H, panelH3), with the majority of crypts showing increased depth and width (FIG.4G; FIG. 4H, panel H3) and significant reduction in Aldh⁺ cells ascompared to untreated mice (FIG. 4J; FIG. 4K, panel K3). Those featureswere aggravated by IGFBP3 administration to STZ-treated B6 mice (FIGS.11D-11G; FIG. 11H, panels H1-H2), with evidences of weight decrease(FIG. 11J), CoSCs loss (FIGS. 11J-11L) and up regulated expression ofCaspase 8 and 9 (FIGS. 11M-11N). Administration of IGF-I i.p inSTZ-treated B6 mice only partially improved mucosa morphology increasedthe number of normal crypts, which remained abnormal (FIG. 11D), andonly partially restored the number of Aldh⁺ cells (FIG. 11G; FIG. 11H,panels H1-H2).

Treatment of Long-Standing T1D with Simultaneous Pancreas-KidneyTransplantation (SPK) Reverts Clinical and Morphological Features of DE

The gold standard treatment for long-standing T1D is SPK, which affordsstable glycometabolic control, near-normalize risk factors and prolongedsurvival (Table VI)(Fiorina et al., 2004; Fiorina et al., 2005; Folli etal., 2010; Secchi et al., 1998; Smets et al., 1999).

TABLE VI Restoration of both normoglycemia and normal renal function inSPK is associated with stable glucose/lipid metabolism and bloodpressure control over time at up to 8 years of follow-up as compared toK + T1D (data are shown at 8 years of follow-up). T1D + ESRD SPK K + T1DParameters (n = 60) (n = 30) (n = 30) P value eGFR <15 65.6 ± 20.2* 61.8 ± 25.2^(§) *, ^(§)<0.0001 (ml/min/1.73 m²) HbA1c (%)  8.4 ± 1.55.4 ± 0.3*  7.5 ± 1.4^(§) *<0.0001; ^(§)<0.001 EIR (UI) 37.4 ± 2.3 0*26.0 ± 7.0^(§) *<0.0001; ^(§)0.001  TG (mg/dl) 162.5 ± 92.7 90.4 ± 23.0*147.1 ± 98.0^(§)  *0.01; ^(§)0.04 Chol (mg/dl) 201.0 ± 45.7 185 ± 27.2 191.1 ± 41.1  Ns LDL (mg/dl) 116.3 ± 40.3 119.5 ± 34.0  97.8 ± 2.1  NsHDL (mg/dl)  48.1 ± 14.4 51.4 ± 4.1  43.13 ± 5.7   Ns Systolic BP 146.3± 18.7 133.1 ± 14.2*  140.1 ± 15.7^(§)  0.03; ^(§)0.04 Diastolic BP 83.7± 8.3 79.1 ± 9.2  78.3 ± 9.2  Ns Abbreviations: T1D, type 1 diabetes;ESRD, end stage renal disease; SPK, simultaneous kidney-pancreastransplantation; K + T1D, kidney transplantation alone in type 1diabetes; eGFR, estimated glomerular filtration rate; HbA1c, glycatedhemoglobin; EIR, exogenous insulin requirement; TG, triglycerides; Chol,total cholesterol; LDL, low density lipoprotein; HDL, high densitylipoprotein; BP, blood pressure; UI, International Unit.

However, individuals with T1D+ESRD are also treated with kidneytransplantation alone but remain diabetic (K+T1D)(Fiorina et al., 2001).A significant improvement in gastrointestinal symptoms was evident overtime after SPK in inventors' cohort of transplanted individuals, whilethe K+T1D group did not report any benefit (FIGS. 12A-12C), suggestingthat DE is reversible.

Treatment of Long-Standing T1D with SPK Re-Establishes Intestinal MucosaMorphology and Local Self-Renewal Properties

Analysis of intestinal mucosa samples showed a significant recovery inthe structure of the epithelial compartment, with compensatoryepithelial hyperplasia in the SPK group (FIG. 12D, panels D1-D2).Recovery of normal crypt histology and number was evident in the SPKgroup when long-standing T1D was successfully treated while none ofthese features were evident in individuals who received kidneytransplant only and remained diabetic (FIG. 12D, panels D1-D2).Epithelial cell proliferation (MIB1⁺ cells) increased after SPK overtime as compared to baseline and to K+T1D at each timepoint (FIG. 4L,FIG. 4M, panels M1-M2), with near-normalization of intestinalmorphology, epithelial renewal and neural features (FIG. 12E, panelsE1-E2; FIG. 12F; FIG. 12G, panels G1-G2; FIG. 12H-12I, FIG. 12J, panelsJ1-J2; FIG. 12K). This demonstrates that treatment of long-standing T1Dwith SPK promoted recovery of intestinal epithelial repair and ofself-renewing properties.

Treatment of Long-Standing T1D Promotes Restoration of CoSCs

Treatment of long-standing T1D with SPK is associated with an increasein Aldh⁺ cells (FIG. 4N, FIG. 4O, panels O1-O2) and EphB2⁺ expression inthe intestinal crypt (FIG. 4P; FIG. 4Q, panels Q1-Q2) and nearlynormalizes the percentage of EphB2^(hi+), EphB2⁺hTERT⁺ andEphB2^(hi+)LGR5⁺ cells in isolated intestinal crypts as compared tobaseline (FIGS. 5A-5C). CoSC marker expression (FIGS. 5D-5G) andgrowth/morphology of mini-guts obtained from SPK individuals were nearlynormalized as well (FIG. 5H; FIG. 13, panels A1-A6). Transcriptomeanalysis revealed that SPK nearly restored the expression of stem celland CoSC markers and of pathways involved in preserving CoSCs (FIG. 5I,FIG. 13B, Table VII).

TABLE VII List of up and down-regulated stem cell target genesidentified by transcriptomic profiling in SPK as compared to T1D + ESRDfreshly isolated colonic crypts (at least p < 0.05). Down-regulatedgenes Up-regulated genes DVL1 ACTC1 APC CCND2 WNT1 BTRC SOX1 SOX2 ACANCOL1A1 COL2A1 BMP3 CCNE1 CDK1 CXCL2 CD8B MME DLL3 HDAC2 JAG1 DTX2 FGF2GDF3 ISL1 MSX1 MYO1 GJA1 RB1 h-TERT NCA1 SIGMAR1 PDX1 DHH BGLA PAbbreviations: EGF, epithelial growth factor; FGF, fibroblast growthfactor, BMP, bone morphogenetic protein.

It is concluded that treatment of long-standing T1D with SPK promotesrestoration of CoSCs.

Treatment of Long-Standing T1D with SPK Restores Circulating IGF-I andIGFBP3

Broad proteomic analysis and targeted immunoassay, revealed anear-normalization of IGFBP3 and IGF-I serum levels after SPK (FIGS.5J-5K) in association with a nearly re-established expression of IGF-IR(FIG. 13C). These results were not evident in the K+T1D group, whoshowed low levels of IGF-I (FIG. 5J) and IGF-IR expression (FIG. 13C)and only a partial recovery in their IGFBP profile (FIG. 13D). Asignificant correlation between IGFBP3 serum levels and intestinalsymptoms in both SPK and K+T1D groups, but more evident in the latter,confirmed that the restoration of IGFBP3 levels is associated with animprovement in diabetic enteropathy (FIGS. 5L-5M; FIGS. 14A-14G).Treatment of long-standing T1D with SPK ameliorates diabetic enteropathyvia a glucose-associated restoration of the circulating IGF-I/IGFBP3dyad.

The Ecto-TMEM219 Recombinant Protein Abrogates IGFBP3-Mediated Mini-GutDestruction In Vitro and Preserves CoSCs In Vivo in a Murine Model ofDE.

In order to further demonstrate the IGFBP3-mediated detrimental effectson CoSCs, the inventors generated a recombinant protein based on theTMEM219 extracellular domain (ecto-TMEM219). Addition of ecto-TMEM219(2:1 molar ratio with IGFBP3) to crypts obtained from CTRL and culturedwith IGFBP3 abrogated the pro-apoptotic effect of IGFBP3 on mini-gutsand preserved the regenerative properties of crypts to generatemini-guts (FIG. 6A). The expression of CoSC signature markers, EphB2 andLGR5, significantly recovered in mini-guts cultured with IGFBP3 andecto-TMEM219, emphasizing a favorable effect in preserving CoSCs (FIG.6B), which was also confirmed in high glucose-cultured mini-guts (FIG.6A). Moreover, Analysis of Caspase 8 and 9 by RT-PCR documented a netdecrease in their expression when ecto-TMEM219 was added toIGFBP3-cultured mini-guts as compared to IGFBP3 alone (FIGS. 6C-6D). Theinventors then treated STZ-B6 mice with ecto-TMEM219 and observedimproved mucosa morphology with recovered number, depth and width ofcrypts (FIGS. 6E-6G). Administration of ecto-TMEM219 was associated withan increase in mice body weight as compared to STZ-treated B6 (FIG. 6H),with significant regain of CoSCs (FIGS. 6I-6K), a decreased expressionof caspase 8 and 9 (FIGS. 6L-6M) and a re-establishment of circulatingIGFBP3 levels (FIG. 6N).

Discussion

Diabetic enteropathy represents a clinically relevant complication inindividuals with T1D, as it is associated with lower quality of life,malnutrition and malabsorption (Bytzer et al., 2002; Faraj et al., 2007;Talley et al., 2001). Particularly, in individuals with long-standingT1D (T1D+ESRD), intestinal disorders occur with high frequency andseverity (Cano et al., 2007; Wu et al., 2004), resulting in body massloss and cachexia (Pupim et al., 2005), indicating that enteropathy isan important complication of long-standing T1D (Atkinson et al., 2013;Pambianco et al., 2006). Inventors' results demonstrate that individualswith long-standing T1D experienced severe intestinal disorders (TableVIII) and that these clinical conditions are associated with alterationsof the intestinal mucosa, with reduced proliferation of intestinalepithelial cells and with signs of neural degeneration.

TABLE VIII Overview of results of diabetic enteropathy assessment inT1D + ESRD individuals as compared to CTRL and SPK. T1D + ESRD SPK vs.vs. Results CTRL T1D + ESRD Metabolic Evaluation Glucose metabolism −−−+++ Lipid metabolism −− + Blood pressure control −− + IntestinalSymptoms Diarrhea −−− +++ Abdominal pain −−− +++ Constipation −−− ++Anorectal Manometry Resting tone = = Contracting tone −− = Reflexresponse −− = Urgency volume −− ++ Mucosa Epithelial RenewalProliferation −−− +++ Differentiation −−− +++ Neural Regeneration Nerves−−− +++ Schwann cells −−− +++ Colonic Stem Cell Turnover Colonic stemcells −−− +++ Crypt growth −−− +++ Arbitrary unit: +++ (highimprovement); ++ (mild improvement); + (slight improvement); = noimprovement; −−− (severe worsening); −− (mild worsening), − (slightworsening). Evaluations were performed as follows: T1D + ESRD vs. CTRL,SKP vs. T1D + ESRD, K + T1D vs. SKP. Abbreviations; T1D, type 1diabetes; ESRD, end stage renal disease; CTRL, healthy subjects; SPK,simultaneous kidney-pancreas transplantation.

Similar features have also been reported in rodent models of T1D and DE(Domenech et al., 2011). Inventors' data, for the first time, link DE toa defect in CoSCs and implicate IGFBP3 as having an important role inthe maintenance of intestinal epithelium homeostasis. Whilehyperglycemia is a prominent feature of T1D, inventors' in vitro studiessuggest that this feature cannot fully explain DE and that circulatingfactors may play an important role. Proteomic analysis led to theidentification of IGF-I as an enterotrophic factor involved in thehomeostasis of CoSCs. The inventors then confirmed that IGF-I and IGFBP3control CoSCs and that this axis is dysfunctional in long-standing T1D.Inventors' data indicate that IGF-I acts as a circulating enterotrophicfactor that promotes crypt growth and controls CoSCs through IGF-IR,while IGFBP3 can block IGF-I signaling by binding circulating IGF-I andreducing its bioavailability. In addition, and most importantly, theinventors showed that IGFBP3 acts through a pro-apoptoticIGF-I-independent mechanism on CoSCs, which the inventors demonstratedexpress TMEM219 (the IGFBP3 receptor), thereby inducing the failure ofmini-gut growth. This latter effect is Caspase 8 and 9-mediated andTMEM219-dependent; indeed, the absence of the IGFBP3 receptor (TMEM219)on CoSCs greatly diminished high glucose-associated CoSC injuries. T1Dtogether with starvation and cachexia are characterized by lowcirculating IGF-I levels (Bondy et al., 1994; Giustina et al., 2014) dueto reduced hepatic IGF-I release, which is controlled and stimulated byendogenous insulin (Le Roith, 1997; Sridhar and Goodwin, 2009). Moreimportantly, hyperglycemia appeared to have a direct effect on hepaticsynthesis and release of IGFBP3. IGFBP3 may thus act as a hepatichormone that reduces intestinal absorptive capacity duringhyperglycemia. Interestingly, SPK provided a proof of concept to theinventors' hypothesis and supported their findings regarding theexistence of circulating factors that control CoSCs. The strikingimprovement of clinical and functional features of DE that the inventorsobserved in their study, associated with replenishment of the CoSCs andwith restoration of the circulating IGF-I and IGFBP3, strengthensinventors' hypothesis. Finally, the newly generated ecto-TMEM219recombinant protein improved DE in diabetic mice in vivo and restoredthe ability of mini-guts to grow normally in vitro, thus confirming therole of IGFBP3 in controlling CoSCs and paving the way for a novelpotential therapeutic strategy. In summary, inventors' study shows thatan IGFBP3-mediated disruption of CoSCs linked to hyperglycemia isevident in DE. The inventors suggest that circulating IGF-I/IGFBP3represent a hormonal dyad that controls CoSCs and a novel therapeutictarget for individuals with intestinal disorders, in particular causedby diabetes mellitus of long duration (Bondy et al., 1994; Bortvedt andLund, 2012; Boucher et al., 2010).

Example 2

Materials and Methods

Patients and Study Design

60 individuals with T1D+ESRD registered on the waiting list forsimultaneous pancreas-kidney transplantation (SPK) matched for (age 41to 43 years old), gender, and duration of T1D (29.4±1.8 years) wereenrolled in the study. 20 subjects affected by type 1 diabetes (T1D)from 10 to 20 years were enrolled as well. 20 healthy subjects matchedfor age and gender (CTRL), with normal renal function and normalglycometabolic parameters, were studied as well. T1D+ESRD subjects wereall on intensive insulin treatment at the time of enrollment in thestudy, while the CTRL group was not being administered any medication.All T1D+ESRD subjects were on the same treatment as antiplatelet therapy(ASA) and anti-hypertension (angiotensin-converting-enzyme inhibitors),while 40 out of 60 received statins when enrolled in the study. Subjectswith clear signs of inflammatory bowel diseases as well as celiacdisease were not enrolled.

T1D+ESRD individuals were followed up for 8 years (mean follow-up:8.6±1.1 years) after receiving either SPK (n=30) or K+T1D (n=30)transplantation according to the macroscopic surgical evaluation at thetime of transplantation. Individuals taking an oral anticoagulant agentwere not included. SPK individuals were all insulin-independent for theentire follow-up period, whereas K+T1D individuals were on intensivesubcutaneous insulin therapy. All subjects provided informed consentbefore study enrollment. Studies not included in the routine clinicalfollow-up were covered by an appropriate Institutional Review Boardapproval (Enteropatia-trapianto/01 Secchi/Fiorina).

IGFBP3 Assessment in Urine and Serum

Serum was collected from 3 ml of fresh blood after centrifugation. Urinesamples were collected fresh, centrifuged and stored at −80° C. IGFBP3levels of all groups of subjects were assessed in frozen samples ofserum and urine using commercially available ELISA kits, according tothe manufacturer's instructions (R&D).

Statistical Analysis

Correlation analysis and graphs were performed using Prism Graphpadsoftware. Correlation analysis included assessment of IGFBP3 levels inserum vs. urine of individuals evaluated, IGFBP3 serum levels vs.estimated glomerular filtration rate (eGFR). Statistical significancewas considered when p value was <0.05.

Measurement of Renal Function and Glycometabolic Parameters

MDRD formula was used to assess estimated glomerular filtration rate(eGFR) in ml/min/m2. Blood tests included assessment of Creatinine,blood glucose, glycated hemoglobin in all subjects enrolled in the studyfocusing on comparing CTRL with T1D individuals and individuals withlongstanding T1D (T1D+ESRD).

Results

Serum IGFBP3 Levels Correlates with Urinary IGFBP3 Levels

Analysis of serum and urine levels of IGFBP3 in all subjects enrolled inthe study documented a significant increase of both serum (FIG. 7A) andurine (FIG. 7B) levels of IGFBP3 in T1D+ESRD subjects as compared toCTRL and to a lesser extent to T1D individuals. A significantcorrelation between urine levels and serum levels of IGFBP3 was observedin all subjects evaluated (FIG. 7C). Higher levels of serum IGFBP3correlate with higher levels of urinary IGFBP3. In order to exclude thatthis might be related to renal function, a correlation between IGFBP3serum levels and renal function (eGFR) was performed. IGFBP3 serumlevels were significantly higher in subjects with ESRD (eGFR<15ml/min/m2) (FIG. 7D). However, subjects with an eGFR>15 ml/min/m2, thusnot affected by ESRD, regardless the presence and history of T1D, didnot show any statistically significant correlation between eGFR andIGFBP3 serum levels (FIG. 7E). Considering the correlation betweenIGFBP3 urinary vs. serum levels in CTRL and comparing their means andmedians values within the 25° and 75° percentiles, inventors may set upa range for urinary IGFBP3 as following:

-   -   <350 pg/ml: normal levels (levels observed in healthy subjects)    -   350-500 pg/ml: altered levels (levels observed in T1D with a        history of disease <5 years)    -   >500 pg/ml: indicative of enteropathy (levels observed in        long-standing T1D, T1D subjects with other T1D complications,        history of T1D >5 years).

The inventors can also identify a normal range of urinary IGFBP3 levels(<350 pg/ml) by considering its correlation with serum IGFBP3 levels asrepresented in the gray area in FIG. 7: F.

Example 3

Five individuals with long-term inflammatory bowel disease (IBD) wereenrolled and screened for peripheral levels of IGFBP3, IGF-1 and theratio of the IGFBP-3/IGF-1, according to the same method described abovefor the analysis of diabetic samples.

It was found that while IGFBP3 was slightly increased, the levels ofIGF1 were severely reduced with an overall alteration of IGFBP3/IGF1ratio (FIG. 18). Thus, in inflammatory bowel disease, a large amount ofIGFBP3 is free and available to exert its toxic effect on the intestinalstem cells.

Consequently, an inhibitor of IGFBP3 is also beneficial for thetreatment and/or prevention of inflammatory bowel diseases.

Example 4

Material and Methods

Patients and Study Design

Sixty serum samples from individuals with type 1 (T1D), with T1D of long(>15 years) duration (long-standing T1D) and healthy volunteers (CTRL)matched for age and gender were obtained from blood collection at theSan Raffaele Hospital. Twenty serum samples from individuals screenedpositive for islets Autoantibodies test were collected at thecollaborating site of Gainsville (Florida). 235, 200 and 81 serumsamples from normal glucose tolerant (NGT), impaired glucose tolerant(IGT) and type 2 diabetes (T2D) individuals were collected fromUniversity of Pisa (Italy) under the Genfiev protocol study. NGT, IGT,and T2D were determined based on the results of OGTT test according tothe ADA 2003 criteria.

T1D and long-standing T1D subjects were all on intensive insulintreatment at the time of enrollment in the study, while the CTRL groupwas not being administered any medication. All T1D subjects were on thesame treatment as antiplatelet therapy (ASA) and anti-hypertension(angiotensin-converting-enzyme inhibitors). Concomitant treatment,inclusion and exclusion criteria have been already described (DiabetesCare 2015) and reported at the following websiteclinicaltrials.gov/ct2/show/record/NCT00879801?term=GENFIEV.

All subjects provided informed consent before study enrollment. Studiesnot included in the routine clinical follow-up were covered by anappropriate Institutional Review Board approval(Enteropatia-trapianto/01 Secchi/Fiorina).

Pancreatic Islets

The human islets used in this study were isolated from cadaveric organdonors according to the procedure already described (Petrelli et al.,2011) in conformity to the ethical requirements approved by the NiguardaCà Granda Ethics Board. Briefly, islets were isolated using theautomated method already described (D'Addio et al., 2014). Two types ofenzymes were used: collagenase type P (1-3 mg/ml) and liberase (0.5-1.4mg/ml) (Roche, Indianapolis, Ind., USA). Islets were purified bydiscontinuous gradient in syringes (density gradient: 1,108; 1,096;1,037: Euroficoll, (Sigma-Aldrich, Milan, Italy), or by continuousgradient with refrigerated COBE processor as previously described (Nanoet al., 2005). After isolation, islets were cultured at 22° C. in ahumidified atmosphere (5% CO₂), in M199 medium (Euroclone, Celbio,Milan, Italy) or CMRL (Mediatech, Cellgro, Va., USA) supplemented with10% FCS, 100 μ/ml penicillin, 100 μg/ml streptomycin sulphate(Euroclone, Celbio) and 2 mmol/1 glutamine (Mediatech, Cellgro, Va.,USA). In vitro characterization and culture of islets was performed onislet material processed within 72 h after isolation. Islets werecultured in CMRL 10% FCS, supplemented with 100 μg/ml streptomycinsulphate (Euroclone, Celbio) and 2 mmol/1 glutamine (Mediatech, Cellgro,Va., USA) with a glucose concentration of 5 mM for 4 days.

Murine islets were kindly provided by Prof. James Markmann(Transplantation Unit, Department of Surgery, Massachusetts GeneralHospital, Harvard Medical School, Boston) (Ben Nasr et al., 2015b;Vergani et al., 2010). Pancreatic islets were isolated from C57B16/Jmice by collagenase digestion followed by density gradient separationand then hand-picking, as described previously (Forbes et al., 2010).Islets were then plated and cultured in RPMI 1640 medium supplementedwith L-glutamine, penicillin and 10% as already described, with aglucose concentration of 5 mM for 4 days.

Beta Cell Lines

Mouse βTC3 and αTC1 cells were kindly provided by Carla Perego,University of Milan, with the permission of Prof. Douglas Hanahan(Department of Biochemistry and Biophysics, University of California,San Francisco, Calif.)(Di Cairano et al., 2011). βTC3 were cultured inRPMI 1640 medium (Sigma) containing 0.1 mM glutamic acid andsupplemented with 0.7 mM glutamine as described (Di Cairano et al.,2011). The glucose concentration was 11 mM for cell lines.

Pathology and Immunohistochemistry

Samples were fixed in buffered formalin (formaldehyde 4% w/v and acetatebuffer 0.05 M) and routinely processed in paraffin wax. 3 μm-thicksections of each enrolled case were stained with Hematoxylin & Eosin(H&E) for morphological evaluations. For immunohistochemistry, 3μm-thick sections were mounted on poly-L-lysine coated slides,deparaffinized and hydrated through graded alcohols to water. Afterantigen retrieval, performed by dipping sections in 0.01 M citratebuffer, pH 6 for 10 minutes in a microwave oven at 650 W as well asendogenous peroxidase activity inhibition, performed by dipping sectionsin 3% hydrogen peroxide for 10 minutes, incubation with primaryantibodies was performed at 4° C. for 18-20 hours, followed by theavidin-biotin complex procedure. Immunoreactions were developed using0.03% 3,3′diaminobenzidine tetrahydrochloride, and then sections werecounterstained with Harris' hematoxylin. The following antibodies wereused: insulin (Dako, A0564), anti-IGFBP3 primary antibody (polyclonal,1:50 dilution, Sigma Aldrich HPA013357) and anti-TMEM219 primaryantibody (polyclonal, 1:100, Sigma HPA059185). These antibodies wereimmunohistochemically tested in pancreatic tissues of healthy subjects,B6 and NOD mice and in liver biopsies of patients with T1D/T2D, islettransplanted patients who did not achieve insulin independence. Tissueswithout pathological findings were used as controls. All of these tissuesamples came from the files stored at the Unit of Pathology of theDepartment of Biomedical, Biotechnological, and Translational Sciences,University of Parma, Parma, Italy. The immunostaining intensity wasgraded as 1 (mild), 2 (moderate), and 3 (strong), while its diffusion as1 (focal), 2 (zonal), and 3 (diffuse).

Immunofluorescence

Immunofluorescence samples were observed using a confocal system (LeicaTCS SP2 Laser Scanning Confocal). Images were acquired in multitrackmode, using consecutive and independent optical pathways. The followingprimary antibodies were used for staining of human tissues/cells: mousemonoclonal anti-caspase cleavage product of cytokeratin 18 M30 (cloneM30, Hoffmann-LaRoche, Basel, Switzerland), rabbit polyclonal IGFBP3(1:250, Sigma, HPA013357), rabbit polyclonal TMEM219 (1:250, Sigma,HPA059185) and Guinea Pig polyclonal insulin (1:50, DAKO, A0564). Thefollowing primary antibodies were used for staining of murinetissues/cells: rabbit polyclonal IGFBP3 (1:250, Sigma, SAB4501527), goatpolyclonal TMEM219 (1: 50, Santa Cruz, 244405), Guinea Pig polyclonalinsulin (1:50, DAKO, A0564). The following secondary antibodies wereused for staining of human tissues/cells: donkey anti-rabbit FITC(Jackson) and donkey anti-guinea pig TRITC (Jackson). The followingantibody was used for staining of murine tissues/cells: donkey anti-goatFITC (Jackson).

Human and murine pancreatic islets co-cultured with/without IGFBP3 (LifeTechnologies, 10430H07H5), with/without ecto-TMEM219 (generated by us incollaboration with Genscript, 130 ng/ml), with/without high glucose (20mM Glucose), with/without IFN-γ and IL-1β (R&D Systems, Minneapolis,Minn. 201-LB-005, 2 ng/ml and PeProTech, 300-02, 1,000 U/ml), werestained with TMEM219, insulin and M30 for immunofluorescence forco-localization studies. Murine beta cells co-cultured in the sameconditions as pancreatic islets, were fixed in 10% neutral buffered for30 min, washed with PBS three times and permeabilized with PBS-BSA 2%triton ×100 0.3% for 20 min, blocked with serum 10%, and finallyincubated with primary antibodies over-night at 4° C. and subsequentlylabeled with fluorescent secondary antibodies for 2 hour at roomtemperature. Primary and secondary antibodies were selected as describedabove.

Islets and Beta Cells In Vitro Studies and Characterization

Culturing Conditions

Human and murine islets were cultured at different glucose concentration(5 mM, 20 mM, Sigma), with/without inflammatory stimuli/cocktail(IFN-γ+IL-1β, 2 ng/ml R&D Systems and 1,000 U/ml Peprotech,respectively), with/without IGFBP3 (Life Technologies, 50 ng/ml),with/without ecto-TMEM219 (130 ng/ml, see Recombinant proteins andinterventional studies) and islets were collected for immunofluorescencestudies, RNA extraction, apoptosis detection, and protein analysis.Supernatants were collected for assessment of insulin, IGFBP3 and IGF-Isecretion.

β-TC were cultured as previously described, with/without inflammatorystimuli/cocktail (IFN-γ+IL-1β), with/without IGFBP3, with/withoutecto-TMEM219 (see Recombinant proteins and interventional studies) andcells were collected as for islets studies.

Immunoblotting

Total proteins of intestinal bioptic samples were extracted in Laemmlibuffer (Tris-HCl 62.5 mmol/1, pH 6.8, 20% glycerol, 2% SDS, 5%b-mercaptoethanol) and their concentration was measured. 35 mg of totalprotein was electrophoresed on 7% SDS-PAGE gels and blotted ontonitrocellulose (Schleicher & Schuell, Dassel, Germany). Blots were thenstained with Ponceau S. Membranes were blocked for 1 h in TBS (Tris [10mmol/l], NaCl [150 mmol/l]), 0.1% Tween-20, 5% non-fat dry milk, pH 7.4at 25° C., incubated for 12 h with a rabbit polyclonal IGFBP3 antibody(Sigma, HPA013357) diluted 1:250, or goat polyclonal TMEM219 (Santa CruzBiotechnology, 244404 or 244405) diluted 1:200 or with a monoclonalmouse anti-b-actin antibody (Santa Cruz Biotechnology) diluted 1:500 inTBS-5% milk at 4° C., washed four times with TBS-0.1% Tween-20, thenincubated with a peroxidase-labeled mouse anti-rabbit IgG secondaryantibody (DAKO) (for IGFBP3) or rabbit anti-goat (for TMEM219) or rabbitanti mouse for b-actin, diluted 1:1000 (Santa Cruz Biotechnology) inTBS-5% milk, and finally washed with TBS-0.1% Tween-20. The resultingbands were visualized using enhanced chemiluminescence (SuperSignal;Pierce, Rockford, Ill., USA).

qRT-PCR Analysis

RNA from purified intestinal crypts was extracted using Trizol Reagent(Invitrogen), and qRT-PCR analysis was performed using TaqMan assays(Life Technologies, Grand Island, N.Y.) according to the manufacturer'sinstructions. The normalized expression values were determined using theΔΔCt method. Quantitative reverse transcriptase polymerase chainreaction (qRT-PCR) data were normalized for the expression of ACTB, andΔCt values were calculated. Statistical analysis compared geneexpression across all cell populations for each patient via one-wayANOVA followed by Bonferroni post-test for multiple comparisons betweenthe population of interest and all other populations. Statisticalanalysis was performed also by using the software available RT² profilerPCR Array Data Analysis (Qiagen). For two groups comparison Student ttest was employed. Analysis was performed in triplicates before/after 3days of culture. Below are reported the main characteristics of primersused for human genes:

Gene Refseq Band Size Reference Symbol UniGene # Accession # (bp)Position INS Hs.272259 NM_000207.2 126 252 IGF-IR Hs.643120 NM_000875.364 2248 TMEM219 Hs.460574 NM_001083613.1 60 726 LRP1 Hs.162757NM_002332.2 64 656 TGFbR1 Hs.494622 NM_001130916.1 73 646 TGFbR2Hs.604277 NM_001024847.2 70 1981 CASP8 Hs.599762 NM_001080124.1 124 648ACTB Hs.520640 NM_001101 174 730

Below are reported the main characteristics of primers used for murinegenes:

Gene Refseq Band Size Reference Symbol UniGene # Accession # (bp)Position INS Mm.4626 NM_008386.3 80 533 IGF-IR Mm.275742 NM_010513.2 1063901 TMEM219 Mm.248646 NM_026827,1 78 677 LRP1 Mm.271854 NM_032538.2 1042995 TGFbR1 Mm.197552 NM_009370.2 85 90 TGFbR2 Mm.172346 NM_033397.3 1321656 Casp8 Mm.336851 NM_001080126.1 96 1525 GAPDH Mm. 304088 NM_008084.2107 75ELISA Assay

IGF-I and IGFBP3 levels in the pooled sera/plasma of all groups ofsubjects and in all groups of treated and untreated mice were assessedusing commercially available ELISA kits, according to the manufacturer'sinstructions (R&D SG300, and Sigma RAB0235).

Human primary hepatocytes (HEP10™ Pooled Human Hepatocytes, ThermoFisherScientific) were cultured for 3 days in Williams Medium as permanufacturer's instructions at different glucose concentrations: 11 mM,20 mM and 35 mM. Culturing supernatant was collected, and IGFBP3 wasassessed using an IGFBP3 ELISA kit (Sigma, RAB0235) according to themanufacturer's instructions. Collected cells were separated by trypsinand counted with a hemacytometer.

Insulin levels were assayed with a microparticle enzyme immunoassay(Mercodia Iso-Insulin ELISA, 10-1113-01) with intra- and inter-assaycoefficients of variation (CVs) of 3.0% and 5.0%.

Recombinant Proteins and Interventional Studies

Recombinant human IGF-I (Sigma, 13769), 100 ng/ml (IGF-I), recombinanthuman IGFBP3 (Life Technologies, 10430H07H5), 50 ng/ml (IGFBP3),anti-IGF-IR (Selleckchem, Boston, OSI-906), 1 μM/L and ecto-TMEM219(D'Addio et al., 2015), 130 ng/ml were added to islets/cell cultures atday +1 from islets collection/cell culture. Pancreatic islets and betacells were also exposed to complex diabetogenic conditions: 20 mMglucose, the mixture of 2 ng/ml recombinant human IL-1β (R&D Systems,Minneapolis, Minn. 201-LB-005), and 1,000 U/ml recombinant human IFN-γ(PeProTech, 300-02) for 72 h.

IGFBP3 (Reprokine, Valley Cottage, N.Y.) was administered to naive B6mice at 150 μg/mouse/day for 15 days intraperitoneally (i.p.);ecto-TMEM219 was administered in vivo to STZ-treated B6, to 10 weeks oldNOD and to B6 fed a high fat diet (HFD-B6) mice intraperitoneally (i.p.)at a dose of 150 μg/mouse/day for 15 days in STZ-treated B6 and in NOD,and 100 μg/mouse every other day for 8 weeks in HFD-B6 mice.

Animal Studies

Male C57BL/6 (B6) mice and female non-obese diabetic (NOD) mice (4 weeksold and 10 weeks old) were obtained from the Jackson Laboratory, BarHarbor, Me. All mice were cared for and used in accordance withinstitutional guidelines approved by the Harvard Medical SchoolInstitutional Animal Care and Use Committee. B6 mice were rendereddiabetic using a chemical approach with streptozotocin (STZ) injection(225 mg/kg, administered i.p.; Sigma 50130) this model is accepted andvalidated as a model of T1D diabetes (Carvello et al., 2012; Petrelli etal., 2011; Vergani et al., 2013). Diabetes was defined in bothSTZ-treated B6 and NOD as blood glucose levels >250 mg/dL for 3consecutive measures.

To study the onset and progression of T2D, B6 mice (6 weeks old) werehoused in a germfree Animal house in accordance with the Principles ofLaboratory Animal Care (NIH Publication No 85-23, revised 1985) andreceived water and food ad libitum. The study protocol was approved bythe local ethics committee. Mice were fed with either a High Fat Diet(HFD) (DIO diet D12492, 60% of total calories from fat) or a normal-fatdiet (NFD; DIO diet D12450B; 10% of total calories from fat), purchasedfrom Research Diets (Mucedola, Settimo Milanese, Italy). Each group oftreatment or control consisted of 10 animals. After 16 weeks, glycemiawas measured and IV glucose tolerance test (IVGTT) was performed. Thenext day, mice were anaesthetized and then a blood sample was obtainedand pancreas was harvested for histology studies. A portion of thetissue was also snap-frozen and stored in Trizol to perform RT-PCRstudies.

Finally, plasma and serum were collected to perform analysis of IGF-I(IGF-I ELISA kit, R&D MG100), IGFBP3 (IGFBP3 ELISA kit, R&D MGB300) andinsulin levels (Mouse Insulin ELISA kit, Mercodia, 10-1247-01). Bloodglucose was monitored twice per week up to 12 weeks in HFD-B6 in orderto confirm diabetes onset and permanence.

Statistical Analysis

Data are presented as mean and standard error of the mean (SEM) and weretested for normal distribution with the Kolmogorov-Smirnov test and forhomogeneity of variances with Levene's test. The statisticalsignificance of differences was tested with two-tailed t-test and thechi-square (χ2) tests. Significance between the two groups wasdetermined by two-tailed unpaired Student's t test. For multiplecomparisons, the ANOVA test with Bonferroni correction was employed. Alldata were entered into Statistical Package for the Social Science(SPSS®, IBM®, SPSS Inc., Chicago, Ill.) and analyzed. Graphs weregenerated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla,Calif.). All statistical tests were performed at the 5% significancelevel.

Results

IGFBP3 Peripheral Levels are Increased in Pre-Diabetic and DiabeticMice.

In order to identify potential circulating factors that may have a rolein inducing beta cell death, the inventors profiled the serum proteomeof healthy subjects and individuals at risk for T1D, based on thepresence of one or more anti-islets autoantibodies, using an unbiasedproteomic approach. Proteins, which were significantly different(p-value <0.01) in control pool versus individuals at risk for T1D pool,were further submitted to hierarchical clustering analysis. A clearproteomic profile was evident in individuals at risk for T1D (and inovertly T1D as well) as compared to healthy subjects, with more than 50%of the detected proteins segregating in either one group or the other.In particular, the levels of IGF-I binding proteins 3 (IGFBP3) wereincreased in individuals at risk for T1D using an immune-targeted assay(FIG. 19A), and thus preceded the onset of hyperglycemia. Interestingly,IGFBP3 levels were also altered in samples obtained from the GenfievStudy, which enrolled more than 800 individuals, and classified thembased on the results of the OGTT test in three main categories: normalglucose tolerant (NGT), impaired glucose tolerant (IGT) subjects and T2Dindividuals (T2D). The inventors observed that IGFBP3 levels wereincreased in IGT and T2D as compared to NGT subjects, confirming thathigh peripheral levels of IGFBP3 mainly characterized pre-diabeticconditions (FIG. 19B).

To demonstrate the detrimental effect of IGFBP3 on islets and betacells, the inventors first demonstrated that pre-diabetic NOD mice aswell as diabetic NOD mice and streptozotocin-induced diabetic C57BL/6mice (STZ-B6) exhibited increased peripheral IGFBP3 levels as comparedto naïve B6 (FIG. 20A). The inventors then confirmed this in a murinemodel of T2D, the HFD model. C57B16/J (B6) mice fed a high fat diet,which develop T2D in 16 weeks, showed increased levels of peripheralIGFBP3 as compared to B6 mice fed a normal fat diet (FIG. 20B).

Increased IGFBP3 Production by Hepatocytes in Inflamed Environment andin T1D.

Liver is known to be a site of IGFBP3 production. In order to explore ifinflammatory stimuli could influence hepatic IGFBP3 production, theinventors cultured human primary hepatocytes with various cytokines andwith different glucose concentrations (11, 20 and 35 mM) anddemonstrated that IGFBP3 levels in the supernatants increased rapidlyfollowing different pro-inflammatory stimuli and increased glucoselevels (FIGS. 21A-21B).

TMEM219 is Expressed in Human Islets.

In order to evaluate the effect of IGFBP3/TMEM219 axis on islets andbeta cells, the inventors first assessed TMEM219 expression by usingimmunofluorescence and its co-localization with insulin at the confocalmicroscopy (FIGS. 22A, panels A1-A2). Human islets obtained from cadaverdonors whose pancreas were not suitable for organ donation were studied.TMEM219 (green staining) is diffusely expressed within islets andco-localize with insulin (red staining) (FIG. 22A, panels A1-A2). Theinventors further evaluated the expression of the other known receptorsfor IGFBP3 (i.e. LPR1, TGF-βR1 and TGF-βR2) but none appeared expressed(FIG. 22B). The inventors then confirmed TMEM219 expression by usingRT-PCR and WB (FIGS. 22B-22C).

The inventors further proved expression of TMEM219 in murine isletsusing RT-PCR and excluded that of other known IGFBP3 receptors (LRP1,TGF-beta type 1 and TGF-beta type 2) already described in other cellsand models (Baxter, 2013; Forbes et al., 2010) (FIG. 23A). Finally, theinventors made use of the availability of murine beta and alpha celllines (αTC and βTC), and determined by RT-PCR that expression of TMEM219is restricted to beta cells while other islet cells, such as alphacells, do not express it (FIG. 23B) and further confirm TMEM219expression by WB (FIG. 23C). Immunofluorescence staining of TMEM219(green) and its co-localization with insulin was also confirmed on betacell line at the confocal microscope (FIG. 23D).

IGFBP3 Damages a Beta Cell Line In Vitro.

To demonstrate that IGFBP3 targets beta cells within the islets, theinventors cultured a beta cell line (βTC) for 3 days with/withoutIGFBP3. By using a viability/apoptosis assay, the inventors were able todemonstrate a reduced percentage of viable beta cells in IGFBP3-treatedconditions as compared to untreated (FIG. 24A). Interestingly,IGFBP3-treated beta cells also showed a significant increase in caspase8 expression (FIG. 24B) and a reduction in insulin expression by bothimmunofluorescence and RT-PCR (FIG. 24C; FIG. 24D, panels D1-D2; FIG.24E). Interestingly, IGFBP3-induced apoptosis was markedly higher thanthat induced by the pro-inflammatory stimuli IL-1β and IFN-γ (FIGS.24A-24B) and insulin expression and release were only slightly reduced(FIGS. 24C-24E).

IGFBP3 Damages Murine Islets In Vitro.

To further demonstrate the IGFBP3-mediated detrimental effect on islets,the inventors cultured murine islets isolated from C57BL/6 mice for 4days with/without IGFBP3. The appearance of extensive apoptosis asassessed by FACS (Annexin V⁺7AAD⁻) documented that IGFBP3-treated isletsundergo early apoptosis (87±2 vs. 67±2%, p=0.004), associated with anincrease in caspase 8 expression and with a decrease in insulinexpression by RT-PCR (FIGS. 25A-25C).

IGFBP3 Damages Human Islets In Vitro.

The inventors finally confirmed the IGFBP3-mediated detrimental effectsin human islets by demonstrating that in vitro cultured human islets,obtained from cadaver donors whose pancreata were not suitable for organdonation, exposed to IGFBP3 for 4 days underwent greatly to apoptosis(FIG. 26A), showed an increase in caspase 8 expression (FIG. 26B) and anincreased expression of M30 (FIGS. FIG. 8C, panels C1-C2), a marker forapoptosis, associated with a decrease in insulin expression atimmunostaining (FIG. 26D, panels D1-D2) and using RT-PCR (FIG. 26E).

IGFBP3 Injection in C57BL/6 Mice Alters Islet Morphology In Vivo.

In order to confirm that IGFBP3 alters islet morphology, the inventorsinjected recombinant IGFBP3 (Reprokine) in naïve B6 and STZ-treated B6mice (150 μg every day for 15 days). Histology (H&E) analysis ofcollected pancreata demonstrated an increased derangement in islets ofSTZ-B6 IGFBP3-treated mice as compared to islets of naïve and STZ-B6mice, confirmed by scattered insulin expression upon immunostaining(FIG. 27A, panels A1-A6).

The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3-Associated Damagein a Beta Cell Line In Vitro.

To demonstrate that ecto-TMEM219 prevents IGFBP3-associated detrimentaleffects specifically on beta cells, the inventors cultured a beta cellline with IGFBP3 and ecto-TMEM219 and observed that beta cell apoptosiswas greatly reduced by the addition of ecto-TMEM219. The effect was alsoconfirmed by the analysis of caspase 8 expression which appeared reducedin IGI-BP3+ecto-TMEM219-treated beta cells as compared to those culturedwith IGFBP3 only (FIGS. 28A-28B). Insulin expression, as assessed byRT-PCR and immunofluorescence (red), was consistently increased by theaddition of ecto-TMEM219 to IGFBP3-cultured beta cells (FIGS. 28C,panels C1-C3).

The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3-AssociatedDetrimental Effects in Murine Islets In Vitro.

In order to further confirm the therapeutic properties of ecto-TMEM219in preventing IGFBP3-associated damage, the inventors tested the effectof ecto-TMEM219 in cultured murine islet in vitro. The addition ofecto-TMEM219 (2:1 molar ratio with IGFBP3) to isolated C57BL/6 isletsco-cultured with IGFBP3 abrogated the pro-apoptotic effect of IGFBP3.Moreover, caspase 8 expression was significantly reduced in isletscultured with IGFBP3 and ecto-TMEM219 (FIG. 29A). Insulin expression wasincreased by the addition of ecto-TMEM219 to murine islets cultured withIGFBP3 (FIG. 29B), emphasizing a favorable effect of ecto-TMEM219 onpreserving islet function.

The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3 Detrimental Effectson Human Islets In Vitro.

To demonstrate the beneficial effects of ecto-TMEM219 in preventingislets destruction, the inventors cultured human islets with IGFBP3 andecto-TMEM219 for 4 days and the inventors demonstrated a rescue ofIGFBP3-mediated islets damaging by ecto-TMEM219, associated with anincrease of insulin expression and a decrease of caspase 8 expression atRT-PCR (FIGS. 30A-30B).

Interestingly, the co-staining of insulin (red) and M30 (green), amarker for apoptosis, confirmed that insulin-producing cells wereprotected by ecto-TMEM219 during the co-cultured with IGFBP3 (FIG. 30C,panels C1-C3).

The Recombinant Protein ectoTMEM219 Prevents IGFBP3-Associated IsletAlterations.

In order to prove the effect of ecto-TMEM219 in the treatment ofdiabetes, the inventors measured insulin serum levels in STZ-treateddiabetic mice at 8 weeks and observed that insulin was significantlyincreased in those mice that were treated with ecto-TMEM219 (i.p. 150 μgevery other day for 2 weeks) as compared to untreated STZ-B6 (FIG. 31A).Finally, in another model of islet injury in vivo, B6 mice fed with ahigh fat diet (B6-HFD) showed altered blood glucose and insulin levels,while B6-HFD treated with ecto-TMEM219 (i.p. 100 μg every other day for6 weeks) maintained near-normal glucose and insulin levels (FIG. 31B),thus suggesting a curative effect of ecto-TMEM219 in type-1 and type-2diabetes.

Discussion

Type 1 diabetes (T1D) has historically been regarded as a Tcell-mediated autoimmune disease, resulting in the destruction ofinsulin-producing pancreatic beta cells (Bluestone et al., 2010;Eisenbarth, 1986). According to this perspective, an initiating factortriggers the immune response against autoantigens, and the subsequentnewly activated autoreactive T cells target and further destroyinsulin-producing beta cells (Bluestone et al., 2010). Whetherdestruction of beta cells is solely determined by the autoimmune attackor whether other mechanisms such as paracrine modulation, metabolicderegulation and non-immune beta cell apoptosis contribute to T1Dpathogenesis is now a matter of debate (Atkinson and Chervonsky, 2012;Atkinson et al., 2015). Recently, it has been observed thatenvironmental factors (e.g.; viral infections, diet, neonatal exposureto milk and microbiota) may be required to initiate the autoimmuneresponse in T1D (Filippi and von Herrath, 2008; McLean et al., 2015).Thus a new approach to study the pathogenesis of T1D is graduallyemerging (McLean et al., 2015), such that immunological and geneticfactors are no longer considered to be the sole determinant of T1D(Alper et al., 2006; Oilinki et al., 2012). Moreover, the efficacy ofimmunotherapeutic strategies, which have been considered in the lastdecade to be the principal prospect for establishing a cure for T1D, isnow being questioned (Ben Nasr et al., 2015a). While targeting theautoimmune response using an immunosuppressive treatment or apro-regulatory regimen was shown to be satisfactory in rodents, suchstrategies conversely achieved insulin independence in a negligiblenumber of T1D individuals (Atkinson et al., 2015). In addition tounderscoring the difference between animal models and humans, these dataalso shed light on the fact that investigation of the immune responseprimarily examined immune events occurring in the periphery, whilelittle is known with respect to the disease process that occurs withinislets and particularly in beta cells. In this regard, the discovery ofnovel factors involved in the initiation/facilitation of beta cell lossin T1D will be of significant value. Such discoveries may pave the wayfor novel therapeutic approaches capable of halting or delaying the veryfirst phase of the disease. In the present invention it was found thatin individuals at high-risk for T1D and in those with overt T1D, IGFBP3peripheral levels are increased. Interestingly a similar pattern wasalso observed in individuals at risk of developing T2D (IGT, IFG), whereglucose intolerance was already detectable, and in those withestablished T2D, confirming that, despite a different etiology, themediator of beta cell loss, which occurs in both types of diabetes, maybe the same, a betatoxin called IGFBP3. In fact, T1D and T2D are bothcharacterized by a loss of beta cells, which results in a reducedsecretion of insulin, failure to control blood glucose levels andhyperglycemia (Brennand and Melton, 2009; Yi et al., 2014). Despitedifferent etiological mechanisms, either autoimmune response in T1D orinsulin resistance/inflammation in T2D, lead to a progressive reductionof beta cell mass. Several approaches are currently available to treatT1D and T2D, but none of them aims to target beta cell loss, protectfrom beta cell injury and preserve beta cell mass, thus preventingdiabetes onset. IGFBP3 may also be a mechanism to explain thedecompensation observed in patients with T2D, which slowly but steadilylose their beta cell function and stop producing insulin. The chronicIGFBP3 overproduction observed in T2D may favor the destruction of betacells and lead to the failure for instance of oral anti-diabetic agent.The inventors have also observed that the IGFBP3 receptor (TMEM219) isexpressed in murine/human islets, and that its ligation by IGFBP3 istoxic to beta cells, raising the possibility of the existence of anendogenous beta cell toxin (betatoxin) that may be involved in the earlyphase of T1D and in diabetes in general. A non-immunological factor maydetermine islet/beta cell injuries, and facilitate the exposure ofautoantigens to immune cells, thus creating a local inflamed environmentand a sustained immune reaction. Liver has been already documented to bethe primary source for IGFBP3, and its exposure to inflammation and highglucose levels significantly increases IGFBP3 release in thecirculation. As a result, IGFBP3 targets islets and beta cells thusfavoring their damage and loss. Therefore, neutralization ofIGFBP3-mediated beta cell injury through the use of newly generatedinhibitors of IGFBP3/TMEM219 axis, such as recombinant ecto-TMEM219, mayprevent beta cell loss by quenching peripheral IGFBP3, thus blocking itssignaling via TMEM219 and halting/delaying T1D progression (FIGS.32A-32B). This may lead to clinical application in the field of diabetesprevention, resulting in the use of ecto-TMEM219 in individuals athigh-risk for T1D and eventually T2D. Inhibitors of IGFBP3/TMEM219 axismay thus prevent early beta cell injuries associated with the earlyphase of T1D, by inhibiting binding of IGFBP3 to TMEM219 expressed onthe target tissue. Considering its role in preventing early loss of betacells, inhibitors of IGFBP3/TMEM219 axis may also be considered ofbenefit in the early treatment of T2D. Therefore, inhibitors ofIGFBP3/TMEM219 axis may represent a therapeutic strategy that preventdiabetes onset and protect beta cell from loss and damage thus becominga relevant clinical option for individuals at risk of developingdiabetes, both T1D and T2D, and in those with diabetes in the earlystages. Individuals at risk of developing T1D are mainly characterizedby the early detection in the serum of multiple autoantibodies againstislet peptides, which are usually absent in healthy subjects (Ziegler etal., 2013). These individuals are usually relatives (brothers, sisters)of individuals with T1D, but do not have any sign or symptom related toT1D. The probability of progressing to T1D in these subjects within 10years is high, with the majority of them (70%) developing T1D in thenext 15 years, but are often underestimated (Ziegler et al., 2013).Individuals at risk for developing T2D are difficult to identify,especially in the early phase. Prevention consists mainly of lifestylemodifications, which may delay the onset of the disease but could notprevent it (Schwarz et al., 2012). Various screening methods (geneticanalysis, metabolomics profile, obesity and risk factors assessment) toearly detect alterations in glucose metabolism are underway, buttherapeutic agents capable of preventing or protecting from T2D onsetare not available and current options only include anti-diabetic agentsthat control hyperglycemia and delay T2D progression (metformin), oragents that control other risk factors (lipid-lowering and bloodpressure-lowering agents) (Nathan, 2015). Therefore, treatments aimingto reduce the burden of diabetes in the general population, both T1D andT2D, should focus on these high-risk populations. This invention isintended as a new clinical therapeutic agent to be used in individualsat risk for developing diabetes to prevent its onset and in those whoare in the early stages of the disease (new-onset) to protect fromprogression into established diabetes, by counteracting beta cell lossand preserving beta cell mass. Given its role in preventing beta cellsloss and damage, inhibitors of IGFBP3/TMEM219 axis are of use inindividuals at risk for developing T1D or T2D, and in those with thedisease in its early stages.

REFERENCES

-   (1993). The effect of intensive treatment of diabetes on the    development and progression of long-term complications in    insulin-dependent diabetes mellitus. The Diabetes Control and    Complications Trial Research Group. N Engl J Med 329, 977-986.-   Alper, C. A., Husain, Z., Larsen, C. E., Dubey, D. P., Stein, R.,    Day, C., Baker, A., Beyan, H., Hawa, M., Ola, T. O., et al. (2006).    Incomplete penetrance of susceptibility genes for MHC-determined    immunoglobulin deficiencies in monozygotic twins discordant for type    1 diabetes. Journal of autoimmunity 27, 89-95.-   Atkinson, M. A., and Chervonsky, A. (2012). Does the gut microbiota    have a role in type 1 diabetes? Early evidence from humans and    animal models of the disease. Diabetologia 55, 2868-2877.-   Atkinson, M. A., Eisenbarth, G. S., and Michels, A. W. (2013). Type    1 diabetes. Lancet.-   Atkinson, M. A., von Herrath, M., Powers, A. C., and    Clare-Salzler, M. (2015). Current concepts on the pathogenesis of    type 1 diabetes-considerations for attempts to prevent and reverse    the disease. Diabetes care 38, 979-988.-   Barker, N. (2014). Adult intestinal stem cells: critical drivers of    epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 15,    19-33.-   Baxter, R. C. (2013). Insulin-like growth factor binding protein-3    (IGFBP-3): Novel ligands mediate unexpected functions. Journal of    cell communication and signaling 7, 179-189.-   Ben Nasr, M., D'Addio, F., Usuelli, V., Tezza, S., Abdi, R., and    Fiorina, P. (2015a). The rise, fall, and resurgence of immunotherapy    in type 1 diabetes. Pharmacological research: the official journal    of the Italian Pharmacological Society 98, 31-38.-   Ben Nasr, M., Vergani, A., Avruch, J., Liu, L., Kefaloyianni, E.,    D'Addio, F., Tezza, S., Corradi, D., Bassi, R., Valderrama-Vasquez,    A., et al. (2015b). Co-transplantation of autologous MSCs delays    islet allograft rejection and generates a local immunoprivileged    site. Acta diabetologica 52, 917-927.-   Bluestone, J. A., Herold, K., and Eisenbarth, G. (2010). Genetics,    pathogenesis and clinical interventions in type 1 diabetes. Nature    464, 1293-1300.-   Bondy, C. A., Underwood, L. E., Clemmons, D. R., Guler, H. P.,    Bach, M. A., and Skarulis, M. (1994). Clinical uses of insulin-like    growth factor I. Ann Intern Med 120, 593-601.-   Bortvedt, S. F., and Lund, P. K. (2012). Insulin-like growth factor    1: common mediator of multiple enterotrophic hormones and growth    factors. Curr Opin Gastroenterol 28, 89-98.-   Boucher, J., Macotela, Y., Bezy, O., Mori, M. A., Kriauciunas, K.,    and Kahn, C. R. (2010). A kinase-independent role for unoccupied    insulin and IGF-1 receptors in the control of apoptosis. Sci Signal    3, ra87.-   Breault, D. T., Min, I. M., Carlone, D. L., Farilla, L. G.,    Ambruzs, D. M., Henderson, D. E., Algra, S., Montgomery, R. K.,    Wagers, A. J., and Hole, N. (2008). Generation of mTert-GFP mice as    a model to identify and study tissue progenitor cells. Proc Natl    Acad Sci USA 105, 10420-10425.-   Brennand, K., and Melton, D. (2009). Slow and steady is the key to    beta-cell replication. Journal of cellular and molecular medicine    13, 472-487.-   Bytzer, P., Talley, N. J., Hammer, J., Young, L. J., Jones, M. P.,    and Horowitz, M. (2002). GI symptoms in diabetes mellitus are    associated with both poor glycemic control and diabetic    complications. Am J Gastroenterol 97, 604-611.-   Camilleri, M. (2007). Clinical practice. Diabetic gastroparesis. N    Engl J Med 356, 820-829.-   Cano, A. E., Neil, A. K., Kang, J. Y., Barnabas, A., Eastwood, J.    B., Nelson, S. R., Hartley, I., and Maxwell, D. (2007).    Gastrointestinal symptoms in patients with end-stage renal disease    undergoing treatment by hemodialysis or peritoneal dialysis. Am J    Gastroenterol 102, 1990-1997.-   Carlone, D. L., and Breault, D. T. (2012). Tales from the crypt: the    expanding role of slow cycling intestinal stem cells. Cell Stem Cell    10, 2-4.-   Carpentino, J. E., Hynes, M. J., Appelman, H. D., Zheng, T.,    Steindler, D. A., Scott, E. W., and Huang, E. H. (2009). Aldehyde    dehydrogenase-expressing colon stem cells contribute to    tumorigenesis in the transition from colitis to cancer. Cancer Res    69, 8208-8215.-   Carrington, E. V., Brokjaer, A., Craven, H., Zarate, N.,    Horrocks, E. J., Palit, S., Jackson, W., Duthie, G. S., Knowles, C.    H., Lunniss, P. J., et al. (2014). Traditional measures of normal    anal sphincter function using high-resolution anorectal manometry    (HRAM) in 115 healthy volunteers. Neurogastroenterol Motil.-   Carvello, M., Petrelli, A., Vergani, A., Lee, K. M., Tezza, S.,    Chin, M., Orsenigo, E., Staudacher, C., Secchi, A.,    Dunussi-Joannopoulos, K., et al. (2012). Inotuzumab ozogamicin    murine analog-mediated B-cell depletion reduces anti-islet allo- and    autoimmune responses. Diabetes 61, 155-165.-   Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J.    V., and Mann, M. (2011). Andromeda: a peptide search engine    integrated into the MaxQuant environment. J Proteome Res 10,    1794-1805.-   D'Addio, F., La Rosa, S., Maestroni, A., Jung, P., Orsenigo, E., Ben    Nasr, M., Tezza, S., Bassi, R., Finzi, G., Marando, A., et al.    (2015). Circulating IGF-I and IGFBP3 Levels Control Human Colonic    Stem Cell Function and Are Disrupted in Diabetic Enteropathy. Cell    Stem Cell 17, 486-498.-   D'Addio, F., Valderrama Vasquez, A., Ben Nasr, M., Franek, E., Zhu,    D., Li, L., Ning, G., Snarski, E., and Fiorina, P. (2014).    Autologous nonmyeloablative hematopoietic stem cell transplantation    in new-onset type 1 diabetes: a multicenter analysis. Diabetes 63,    3041-3046.-   Di Cairano, E. S., Davalli, A. M., Perego, L., Sala, S., Sacchi, V.    F., La Rosa, S., Finzi, G., Placidi, C., Capella, C., Conti, P., et    al. (2011). The glial glutamate transporter 1 (GLT1) is expressed by    pancreatic beta-cells and prevents glutamate-induced beta-cell    death. The Journal of biological chemistry 286, 14007-14018.-   Domenech, A., Pasquinelli, G., De Giorgio, R., Gori, A., Bosch, F.,    Pumarola, M., and Jimenez, M. (2011). Morphofunctional changes    underlying intestinal dysmotility in diabetic RIP-I/hIFNbeta    transgenic mice. Int J Exp Pathol 92, 400-412.-   Eisenbarth, G. S. (1986). Type I diabetes mellitus. A chronic    autoimmune disease. The New England journal of medicine 314,    1360-1368.-   Faraj, J., Melander, O., Sundkvist, G., Olsson, R., Thorsson, O.,    Ekberg, O., and Ohlsson, B. (2007). Oesophageal dysmotility, delayed    gastric emptying and gastrointestinal symptoms in patients with    diabetes mellitus. Diabet Med 24, 1235-1239.-   Feldman, M., and Schiller, L. R. (1983). Disorders of    gastrointestinal motility associated with diabetes mellitus. Ann    Intern Med 98, 378-384.-   Filippi, C. M., and von Herrath, M. G. (2008). Viral trigger for    type 1 diabetes: pros and cons. Diabetes 57, 2863-2871.-   Fiorina, P., Folli, F., Bertuzzi, F., Maffi, P., Finzi, G.,    Venturini, M., Socci, C., Davalli, A., Orsenigo, E., Monti, L., et    al. (2003). Long-term beneficial effect of islet transplantation on    diabetic macro-/microangiopathy in type 1 diabetic    kidney-transplanted patients. Diabetes Care 26, 1129-1136.-   Fiorina, P., Folli, F., D'Angelo, A., Finzi, G., Pellegatta, F.,    Guzzi, V., Fedeli, C., Della Valle, P., Usellini, L., Placidi, C.,    et al. (2004). Normalization of multiple hemostatic abnormalities in    uremic type 1 diabetic patients after kidney-pancreas    transplantation. Diabetes 53, 2291-2300.-   Fiorina, P., La Rocca, E., Venturini, M., Minicucci, F., Fermo, I.,    Paroni, R., D'Angelo, A., Sblendido, M., Di Carlo, V., Cristallo,    M., et al. (2001). Effects of kidney-pancreas transplantation on    atherosclerotic risk factors and endothelial function in patients    with uremia and type 1 diabetes. Diabetes 50, 496-501.-   Fiorina, P., Venturini, M., Folli, F., Losio, C., Maffi, P.,    Placidi, C., La Rosa, S., Orsenigo, E., Socci, C., Capella, C., et    al. (2005). Natural history of kidney graft survival, hypertrophy,    and vascular function in end-stage renal disease type 1 diabetic    kidney-transplanted patients: beneficial impact of pancreas and    successful islet cotransplantation. Diabetes Care 28, 1303-1310.-   Folli, F., Guzzi, V., Perego, L., Coletta, D. K., Finzi, G.,    Placidi, C., La Rosa, S., Capella, C., Socci, C., Lauro, D., et al.    (2010). Proteomics reveals novel oxidative and glycolytic mechanisms    in type 1 diabetic patients' skin which are normalized by    kidney-pancreas transplantation. PLoS One 5, e9923.-   Forbes, K., Souquet, B., Garside, R., Aplin, J. D., and Westwood, M.    (2010). Transforming growth factor-{beta} (TGF{beta}) receptors I/II    differentially regulate TGF{beta}1 and IGF-binding protein-3    mitogenic effects in the human placenta. Endocrinology 151,    1723-1731.-   Giustina, A., Berardelli, R., Gazzaruso, C., and Mazziotti, G.    (2014). Insulin and GH-IGF-I axis: endocrine pacer or endocrine    disruptor? Acta Diabetol.-   Gracz, A. D., Fuller, M. K., Wang, F., Li, L., Stelzner, M.,    Dunn, J. C., Martin, M. G., and Magness, S. T. (2013). Brief Report:    CD24 and CD44 mark human intestinal epithelial cell populations with    characteristics of active and facultative stem cells. Stem Cells 31,    2024-2030.-   Hsu, S. M., Raine, L., and Fanger, H. (1981). Use of    avidin-biotin-peroxidase complex (ABC) in immunoperoxidase    techniques: a comparison between ABC and unlabeled antibody (PAP)    procedures. J Histochem Cytochem 29, 577-580.-   Hughes, K. R., Sablitzky, F., and Mahida, Y. R. (2011). Expression    profiling of Wnt family of genes in normal and inflammatory bowel    disease primary human intestinal myofibroblasts and normal human    colonic crypt epithelial cells. Inflamm Bowel Dis 17, 213-220.-   Jung, P., Sato, T., Merlos-Suarez, A., Barriga, F. M., Iglesias, M.,    Rossell, D., Auer, H., Gallardo, M., Blasco, M. A., Sancho, E., et    al. (2011). Isolation and in vitro expansion of human colonic stem    cells. Nat Med 17, 1225-1227.-   Kosinski, C., Li, V. S., Chan, A. S., Zhang, J., Ho, C., Tsui, W.    Y., Chan, T. L., Mifflin, R. C., Powell, D. W., Yuen, S. T., et al.    (2007). Gene expression patterns of human colon tops and basal    crypts and BMP antagonists as intestinal stem cell niche factors.    Proc Natl Acad Sci USA 104, 15418-15423.-   Le Roith, D. (1997). Seminars in medicine of the Beth Israel    Deaconess Medical Center. Insulin-like growth factors. N Engl J Med    336, 633-640.-   Levey, A. S., Bosch, J. P., Lewis, J. B., Greene, T., Rogers, N.,    and Roth, D. (1999). A more accurate method to estimate glomerular    filtration rate from serum creatinine: a new prediction equation.    Modification of Diet in Renal Disease Study Group. Ann Intern Med    130, 461-470.-   Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.    (1951). Protein measurement with the Folin phenol reagent. J Biol    Chem 193, 265-275.-   McLean, M. H., Dieguez, D., Jr., Miller, L. M., and Young, H. A.    (2015). Does the microbiota play a role in the pathogenesis of    autoimmune diseases? Gut 64, 332-341.-   Medema, J. P., and Vermeulen, L. (2011). Microenvironmental    regulation of stem cells in intestinal homeostasis and cancer.    Nature 474, 318-326.-   Merlos-Suarez, A., Barriga, F. M., Jung, P., Iglesias, M.,    Cespedes, M. V., Rossell, D., Sevillano,-   M., Hernando-Momblona, X., da Silva-Diz, V., Munoz, P., et al.    (2011). The intestinal stem cell signature identifies colorectal    cancer stem cells and predicts disease relapse. Cell Stem Cell 8,    511-524.-   Munoz, J., Stange, D. E., Schepers, A. G., van de Wetering, M.,    Koo, B. K., Itzkovitz, S., Volckmann, R., Kung, K. S., Koster, J.,    Radulescu, S., et al. (2012). The Lgr5 intestinal stem cell    signature: robust expression of proposed quiescent ‘+4’ cell    markers. EMBO J 31, 3079-3091.-   Muzumdar, R. H., Ma, X., Fishman, S., Yang, X., Atzmon, G., Vuguin,    P., Einstein, F. H., Hwang, D., Cohen, P., and Barzilai, N. (2006).    Central and opposing effects of IGF-I and IGF-binding protein-3 on    systemic insulin action. Diabetes 55, 2788-2796.-   Nano, R., Clissi, B., Melzi, R., Calori, G., Maffi, P., Antonioli,    B., Marzorati, S., Aldrighetti, L., Freschi, M., Grochowiecki, T.,    et al. (2005). Islet isolation for allotransplantation: variables    associated with successful islet yield and graft function.    Diabetologia 48, 906-912.-   Nathan, D. M. (2015). Diabetes: Advances in Diagnosis and Treatment.    Jama 314, 1052-1062.-   Oilinki, T., Otonkoski, T., Ilonen, J., Knip, M., and    Miettinen, P. J. (2012). Prevalence and characteristics of diabetes    among Somali children and adolescents living in Helsinki, Finland.    Pediatric diabetes 13, 176-180.-   Pambianco, G., Costacou, T., Ellis, D., Becker, D. J., Klein, R.,    and Orchard, T. J. (2006). The 30-year natural history of type 1    diabetes complications: the Pittsburgh Epidemiology of Diabetes    Complications Study experience. Diabetes 55, 1463-1469.-   Petrelli, A., Carvello, M., Vergani, A., Lee, K. M., Tezza, S., Du,    M., Kleffel, S., Chengwen, L., Mfarrej, B. G., Hwu, P., et al.    (2011). IL-21 is an antitolerogenic cytokine of the late-phase    alloimmune response. Diabetes 60, 3223-3234.-   Pupim, L. B., Heimburger, O., Qureshi, A. R., Ikizler, T. A., and    Stenvinkel, P. (2005). Accelerated lean body mass loss in incident    chronic dialysis patients with diabetes mellitus. Kidney Int 68,    2368-2374.-   Remes-Troche, J. M., De-Ocampo, S., Valestin, J., and Rao, S. S.    (2010). Rectoanal reflexes and sensorimotor response in rectal    hyposensitivity. Dis Colon Rectum 53, 1047-1054.-   Sato, T., and Clevers, H. (2013). Growing self-organizing mini-guts    from a single intestinal stem cell: mechanism and applications.    Science 340, 1190-1194.-   Schwarz, P. E., Greaves, C. J., Lindstrom, J., Yates, T., and    Davies, M. J. (2012). Nonpharmacological interventions for the    prevention of type 2 diabetes mellitus. Nature reviews.    Endocrinology 8, 363-373.-   Secchi, A., Caldara, R., La Rocca, E., Fiorina, P., and Di Carlo, V.    (1998). Cardiovascular disease and neoplasms after pancreas    transplantation. Lancet 352, 65; author reply 66.-   Smets, Y. F., Westendorp, R. G., van der Pijl, J. W., de Charro, F.    T., Ringers, J., de Fijter, J. W., and Lemkes, H. H. (1999). Effect    of simultaneous pancreas-kidney transplantation on mortality of    patients with type-1 diabetes mellitus and end-stage renal failure.    Lancet 353, 1915-1919.-   Sridhar, S. S., and Goodwin, P. J. (2009). Insulin-insulin-like    growth factor axis and colon cancer. J Clin Oncol 27, 165-167.-   Stange, D. E., and Clevers, H. (2013). Concise review: the yin and    yang of intestinal (cancer) stem cells and their progenitors. Stem    Cells 31, 2287-2295.-   Svedlund, J., Sjodin, I., and Dotevall, G. (1988). GSRS—a clinical    rating scale for gastrointestinal symptoms in patients with    irritable bowel syndrome and peptic ulcer disease. Dig Dis Sci 33,    129-134.-   Talley, N. J., Young, L., Bytzer, P., Hammer, J., Leemon, M., Jones,    M., and Horowitz, M. (2001). Impact of chronic gastrointestinal    symptoms in diabetes mellitus on health-related quality of life. Am    J Gastroenterol 96, 71-76.-   van der Flier, L. G., and Clevers, H. (2009). Stem cells,    self-renewal, and differentiation in the intestinal epithelium.    Annual review of physiology 71, 241-260.-   Vergani, A., D'Addio, F., Jurewicz, M., Petrelli, A., Watanabe, T.,    Liu, K., Law, K., Schuetz, C., Carvello, M., Orsenigo, E., et al.    (2010). A novel clinically relevant strategy to abrogate    autoimmunity and regulate alloimmunity in NOD mice. Diabetes 59,    2253-2264.-   Vergani, A., Fotino, C., D'Addio, F., Tezza, S., Podetta, M., Gatti,    F., Chin, M., Bassi, R., Molano, R. D., Corradi, D., et al. (2013).    Effect of the purinergic inhibitor oxidized ATP in a model of islet    allograft rejection. Diabetes 62, 1665-1675.-   Williams, A. C., Smartt, H., A M, H. Z., Macfarlane, M., Paraskeva,    C., and Collard, T. J. (2007). Insulin-like growth factor binding    protein 3 (IGFBP-3) potentiates TRAIL-induced apoptosis of human    colorectal carcinoma cells through inhibition of NF-kappaB. Cell    Death Differ 14, 137-145.-   Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009).    Universal sample preparation method for proteome analysis. Nat    Methods 6, 359-362.-   Wu, M. J., Chang, C. S., Cheng, C. H., Chen, C. H., Lee, W. C.,    Hsu, Y. H., Shu, K. H., and Tang, M. J. (2004). Colonic transit time    in long-term dialysis patients. Am J Kidney Dis 44, 322-327.-   Yi, P., Park, J. S., and Melton, D. A. (2014). Perspectives on the    activities of ANGPTL8/betatrophin. Cell 159, 467-468.-   Zeki, S. S., Graham, T. A., and Wright, N. A. (2011). Stem cells and    their implications for colorectal cancer. Nature reviews.    Gastroenterology & hepatology 8, 90-100.-   Zhao, J., Yang, J., and Gregersen, H. (2003). Biomechanical and    morphometric intestinal remodelling during experimental diabetes in    rats. Diabetologia 46, 1688-1697.-   Ziegler, A. G., Rewers, M., Simell, O., Simell, T., Lempainen, J.,    Steck, A., Winkler, C., Ilonen, J., Veijola, R., Knip, M., et al.    (2013). Seroconversion to multiple islet autoantibodies and risk of    progression to diabetes in children. Jama 309, 2473-2479.-   Ziskin, J. L., Dunlap, D., Yaylaoglu, M., Fodor, I. K., Forrest, W.    F., Patel, R., Ge, N., Hutchins, G. G., Pine, J. K., Quirke, P., et    al. (2013). In situ validation of an intestinal stem cell signature    in colorectal cancer. Gut 62, 1012-1023.

The invention claimed is:
 1. A method of decreasing apoptosis ofpancreatic beta cells in a subject with type 1 diabetes, comprising:administering an amount of an inhibitor of the IGFBP3/TMEM219 axiseffective to reduce apoptosis of pancreatic beta cells to a subject withtype I diabetes, wherein said inhibitor comprises ecto-TMEM219 (SEQ IDNO: 2).
 2. The method of claim 1, wherein said inhibitor is soluble. 3.The method of claim 1, wherein said inhibitor is pegylated.
 4. Themethod of claim 1, wherein said inhibitor is a host cell geneticallyengineered to express ecto-TMEM219 (SEQ ID NO: 2).
 5. The method ofclaim 1, wherein the subject has early stage Type-1 diabetes.
 6. Themethod of claim 1, wherein said inhibitor of the IGFBP3/TMEM219 axis isadministered as a pharmaceutical composition comprising apharmaceutically acceptable carrier.
 7. The method of claim 6, whereinsaid pharmaceutical composition comprises a second therapeutic agent. 8.The method of claim 7, wherein the second therapeutic agent is selectedfrom the group consisting of: insulin in any form, Pramlintide (Symlin),angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptorblockers (ARBs), Aspirin, Cholesterol-lowering drugs, Metformin,Sulfonylureas, Meglitinides, Thiazolidinediones, DPP-4 inhibitors, GLP-1receptor agonists, and SGLT2 inhibitors.
 9. A method of decreasingapoptosis of pancreatic beta cells in a subject with type 1 diabetes,comprising: administering an amount of an inhibitor of theIGFBP3/TMEM219 axis effective to reduce apoptosis of pancreatic betacells to a subject with type I diabetes, wherein said inhibitorcomprises a polynucleotide coding for ecto-TMEM219 (SEQ ID NO: 2). 10.The method of claim 9, wherein said inhibitor is soluble.
 11. The methodof claim 9, wherein said inhibitor is a vector comprising or expressingsaid polynucleotide.
 12. The method of claim 9, wherein said inhibitoris a host cell comprising said polynucleotide.
 13. The method of claim9, wherein the subject has early stage Type-1 diabetes.
 14. The methodof claim 9, wherein said inhibitor of the IGFBP3/TMEM219 axis isadministered as a pharmaceutical composition comprising apharmaceutically acceptable carrier.
 15. The method of claim 14, whereinsaid pharmaceutical composition comprises a second therapeutic agent.16. The method of claim 15, wherein the therapeutic agent is selectedfrom the group consisting of: insulin in any form, Pramlintide (Symlin),angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptorblockers (ARBs), Aspirin, Cholesterol-lowering drugs, Metformin,Sulfonylureas, Meglitinides, Thiazolidinediones, DPP-4 inhibitors, GLP-1receptor agonists, and SGLT2 inhibitors.
 17. A composition comprising aninhibitor of the IGFBP3/TMEM219 axis, wherein said inhibitor comprisesecto-TMEM219 (SEQ ID NO: 2) or a polynucleotide coding for ecto-TMEM219(SEQ ID NO: 2).
 18. The composition of claim 17, wherein said inhibitoris a vector comprising or expressing said polynucleotide.
 19. Thecomposition of claim 17, wherein said inhibitor is a host cellgenetically engineered to express ecto-TMEM219 (SEQ ID NO: 2).
 20. Thecomposition of claim 17, wherein said inhibitor is a host cellcomprising said polynucleotide.
 21. The composition of claim 17, whereinsaid composition comprises a pharmaceutical composition comprising saidinhibitor and a pharmaceutically acceptable carrier.
 22. The compositionof claim 21, wherein said pharmaceutical composition comprises a secondtherapeutic agent.